A-beta binding polypeptides

ABSTRACT

The invention relates to biparatopic A-beta binding polypeptides and, more specifically, to biparatopic A-beta binding polypeptides comprising at least two immunoglobulin single variable domains binding to different epitopes of A-beta. The invention also relates to specific sequences of such polypeptides, methods of their production, and methods of using them, including methods of treatment of diseases such as Alzheimer&#39;s Disease.

FIELD OF THE INVENTION

The present invention relates to novel beta-amyloid peptide (in thefollowing: “A-beta”) binding polypeptides, the polypeptides comprisingspecific immunoglobulin domains. The invention also relates to nucleicacids encoding such polypeptides; to methods for preparing suchpolypeptides; to host cells expressing or capable of expressing suchpolypeptides; to compositions comprising such polypeptides; and to usesof such polypeptides or such compositions, in particular forprophylactic, therapeutic and diagnostic purposes.

BACKGROUND OF THE INVENTION

Several degenerative neural diseases are caused by the improper foldingor processing of proteins or by prions, both of which result in invasiveneural depositions known as amyloid plaques. The most widely knowndegenerative neural disease is probably Alzheimer's Disease (AD).

The incidence of AD warrants an urgent and unmet medical need: between10 and 40% of all people aged 65 to 85 develop AD. Moreover, thissegment of the population continues to grow exponentially. Therefore,from a humane, as well as from a social and economical point of view, itis imperative to find ways to efficiently diagnose and treat thisdevastating disorder. Concerning treatment, drugs are needed not only toslow or stop the disease progression, but also to restore brain damagethat has already occurred during the initial stages of AD (beforediagnosis). At this moment, neither early-diagnosis nor therapytreatment are efficient.

AD is defined as a dementia that coincides with the presence in thebrain of extracellular amyloid plaques, composed mainly of amyloidpeptides, and by intracellular neurofibrillary tangles (NFT) composedmainly of protein tau.

A primary component of amyloid plaques characteristic of AD is betaamyloid peptide (A-beta), a highly insoluble peptide 39-43 amino acids(aa) in length that has a strong propensity to adopt beta sheetstructures, oligomerize and form protein aggregates. A-beta is producedfrom the A-beta precursor protein (APP) by two proteolytic events. Abeta-secretase activity cleaves APP at the N-terminus of A-beta(“beta-site”) between amino acids Met-671 and Asp-672 (using thenumbering of the 770 aa isoform of APP). Cleavage at the beta-siteyields a membrane-associated APP fragment of 99 aa (C99). A second sitewithin the transmembrane domain of C99 (“gamma site”) can then becleaved by a gamma-secretase to release A-beta. APP can alternatively becleaved within its A-beta region, predominantly at the alpha-secretasecleavage site of APP, to produce a C-terminal APP fragment of 83 aa(C83), which can also be further cleaved by gamma-secretase to produce asmall soluble secreted peptide, p3. This pathway reduces the potentialaccumulation of A-beta.

The intra- and extracellular A-beta adopts a beta-sheet conformation andforms intermediates named ADDL (amyloid derived diffusible ligands) andprotofibrils, finally precipitates in the form of amyloid fibrils whichassemble into amyloid plaques. In these processes, the more hydrophobicA-beta(1-42) peptide (cf. below) is presumed to serve as a nucleatingagent around which the plaques steadily grow.

A number of missense mutations in APP have been implicated in forms ofearly-onset familial AD. All of these are at or near one of thecanonical cleavage sites of APP. Thus, the Swedish double mutation(K670N/M671L) is immediately adjacent to the beta-secretase cleavagesite and increases the efficiency of beta-secretase activity, resultingin production of more total A-beta. Any of three mutations at APPresidue 717, near the gamma-secretase cleavage site, increases theproportion of a more amyloidogenic 42 aa form of A-beta, also namedA-beta(1-42), relative to the more common 40 aa form, A-beta(1-40). Twoadditional mutations of APP have been described which are close but notadjacent to the alpha-site. A mutation (A692G, A-beta residue 21) in aFlemish family and a mutation (E693Q, A-beta residue 22) in a Dutchfamily each have been implicated in distinct forms of familial AD. TheFlemish mutation, in particular, presents as a syndrome of repetitiveintracerebral hemorrhages or as an AD-type dementia. Theneuropathological findings include senile plaques in the cortex andhippocampus, and usually multiple amyloid deposits in the walls ofcerebral microvessels.

Several years ago, the membrane-associated aspartyl protease, BACE (alsocalled memapsin or Asp2) has been shown to exhibit properties expectedof a beta-secretase. This enzyme cleaves APP at its beta-site andbetween Tyr-10 and Glu-11 of the A-beta region with comparableefficiency. A-beta fragments cleaved at this latter site have beenobserved in amyloid plaques in AD and in media of APP-transfected HEK293human embryonic kidney cells. Several groups also observed the presencein the database of an additional aspartyl protease, BACE2 (also calledAsp1), a close homologue of BACE (now also referred to as BACE1). BACE2cleaves APP at its beta-site and more efficiently at sites within theA-beta region of APP, after Phe-19 and Phe-20 of A-beta. These internalA-beta-sites are adjacent to the Flemish APP mutation at residue 21, andthis mutation markedly increases the proportion of beta-site cleavageproduct generated by BACE2. Conservative beta-site mutations of APP thateither increase (the Swedish mutation) or inhibit (M671V) beta-secretaseactivity affect BACE1 and BACE2 activity similarly. BACE2, like BACE1,proteolyzes APP maximally at acidic pH.

Mutations in the APP gene or in the presenilin 1 (PS1) gene (carrying“gamma-secretase” activity) cause early-onset familial AD. Examples ofAPP mutations are the above-mentioned ‘Swedish’ and ‘London’ (717)mutations located respectively near the beta- and gamma-secretasecleavage sites. These mutations increase the formation of A-betapeptides and especially of A-beta(1-42), and thereby increase theformation of amyloid aggregates and plaques. Whereas initially plaqueswere believed to be a major trigger for the development of AD, currentstudies emphasize the role of protofibrils and ADDL as the major toxiccomponents. It is even conceivable that plaques are a mechanism wherebythe neurotoxic peptides are actually rendered biologically inactive.

Further information on neurodegenerative diseases and on the role ofA-beta therein can be taken from Wisniewski & Konietzko (2008), LancetNeurol. 7(9), 805-811, Spires-Jones et al. (2009), Neurobiology ofDisease, 213-220, and Lichlen & Mohajeri (2008), Journal of Neurochem.104, 859-874.

Most current treatments of AD target the acetylcholine deficiency usingacetylcholinesterase inhibitors such as donepezil (Aricept®),galantamine (Reminyl®), and rivastigmine (Exelon®) which are registeredfor the treatment of mild to moderate AD. Donepezil is also approved forsevere Dementia Alzheimer's type (DAT) in the U.S.A. and Canada. Theacetylcholine deficit reflects the degeneration of cholinergic neuronsof the basal forebrain and appears to correlate well with theneuropsychiatric manifestations of the disease. Treatment withacetylcholinesterase inhibitors has some beneficial effects (consistentand significant but modest efficacy on clinical measures of cognitionand global function), but cannot cure or stop the progression of thedisease, as the etiology of the neurodegeneration is left untreated.

Memantine (Axura®, Namenda®, Ebixa®; Merz Pharmaceuticals) is an NMDAreceptor antagonist that showed better outcome in comparison to placeboin the clinical domains cognition, activities of daily living andoverall clinical response in AD patients with moderate to severeAlzheimer's disease. Memantine remains a symptomatic therapy that isapproved for moderate to severe Alzheimer's disease only. It neithercures nor stops the progression of the disease. A combination ofmemantine and acetylcholinesterase inhibitors has been shown to havesuperior efficacy in moderately severe to severe DAT but not in the mildto moderate disease stage.

Some current experimental therapeutic strategies focus on A-beta as atarget. There are three major research lines:

a) The development of small molecules (often peptido-mimetics) namedbeta-sheet breakers, which are designed to interfere with the beta-sheetstructure of amyloid peptide aggregates. It has been demonstrated that astable “beta-sheet breaker”, when administered to a transgenic mousemodel of AD, is able to penetrate the blood brain barrier and reduce thenumber of plaques (Permanne et al. (2002), FASEB J. 16, 860-862). Itremains to be demonstrated whether this approach results in cognitiveprotection and/or restoration.b) The development of small molecules which inhibit the proteolyticprocessing of APP into amyloid peptides. Inhibitors of the beta- orgamma-secretase should efficiently block the formation of A-beta andhence protect the brain from neurotoxic effects of amyloid. Effects onalready existing brain A-beta burden, such as amyloid plaques which haveaccumulated over years, are not expected.c) Passive and active vaccination against A-beta. This research linestarted with the observation by Schenk et al. (1999), Nature 400,173-177, that vaccination of transgenic AD mice with A-beta(1-42)prevented the formation of amyloid plaques. In a first experiment,monthly vaccination of young adult mice (age 6 weeks) essentiallyprevented plaque formation and the concomitant inflammatory reaction inthe brain, i.e. absence of amyloid plaques, of astrocytosis andmicrogliosis. Vaccination starting at a later age, when amyloid plaqueswere already established, resulted in a partial clearance. Subsequently,it was demonstrated that vaccination with A-beta improved the behavioraland memory deficits as measured in the water maze memory tests. Giventhe side-effects of vaccination with the entire A-beta, alternativeshorter peptides have been designed and used to vaccinate transgenicmice. Clinical trials suggested that the active immunization with A-betais therapeutically active, as demonstrated by eliciting plaqueclearance, attenuating plaque-related pathology, decreasing tau levelsand slowing patients' cognitive decline. However, a significant numberof patients developed autoimmune meningoencephalitis, caused primarilyby the infiltration of autoreactive T lymphocytes into the brain inresponse to active immunization (Ferrer et al. (2004), Brain Pathol. 14,11-20; Nicoll et al. (2003), Nat. Med. 9, 448-452; Masliah et al.(2005), Neurology 64, 1553-1562).

As an alternative to active immunization approaches, antibodies directedagainst A-beta may be administered to a patient. Such passiveimmunization approach was shown to be successful in reducing brainA-beta burden in transgenic AD mice (DeMattos et al. (2001), Proc. Natl.Acad. Sci. USA 98, 8850-8855). The underlying mechanisms remain open forspeculation since it was thought unlikely that antibodies could crossthe blood-brain barrier and target the plaques present in brain. Theauthors therefore suggested that the antibody created an ‘A-beta sink’in the plasma which titrated A-beta out of the brain. Subsequently,using gelsolin and ganglioside 1, it was demonstrated that anyA-beta-binding ligand has the potential to reduce amyloid burden intransgenic AD mice without crossing the blood-brain barrier (Matsuoka etal. (2003) J. Neuroscience 23, 29-33). Short-term (24 hours) passiveimmunization appeared to restore cognitive deficits of transgenic ADmice even without affecting the total brain amyloid load (Dodart et al.(2002) Nature Neuroscience 5, 452-457). The result would suggest thatsmaller, still soluble aggregates of A-beta are targeted first by someantibodies, and also that these are the most toxic forms of A-beta.Hence, clearance of proto-fibrillar A-beta could restore memory, atleast in transgenic APP-mice.

The humanized anti-A-beta monoclonal antibody bapineuzumab (an analogueof the anti-A-beta mouse antibody known as “3D6”) has meanwhile enteredclinical trials. However, the first data reported from Phase II trialshowed mixed results: Statistically significant effects on severalefficacy endpoints were observed in ApoE4 non-carriers only.Furthermore, bapineuzumab was well tolerated and safe in ApoE4non-carriers, while in ApoE4 carriers, serious adverse events were morefrequently observed in bapineuzumab-treated patients than in the placeboarm. Moreover, vasogenic edema events have been observed. The inductionof cerebral microhemorrhages has also been described pre-clinically intransgenic APP mice.

Conventional antibodies (containing an Fc part) used in anti-A-betapassive immunizations are suspected to account for the induction ofvasogenic edema or microhemorrhages observed in humans and animalmodels, which are associated with a targeting of cerebral vascularA-beta deposits (Cerebral amyloid angiopathy) leading to microbleedingsvia ADCC and/or CDC (Wilcock, D M, Colton, C A, CNS Neurol. Disord. DrugTargets (2009) Vol. 8(1):50-64). Finally, the binding affinity of about2.5 nM of this antibody, as measured by Biacore, is assumed to be toolow to induce an effective “peripheral sink effect”.

Another anti-A-beta antibody, solanezumab (humanized antibody m266;LY-2062430), has also entered clinical testings. The maximal plaque loadreduction that could be achieved was published to be about 60%. Inaddition, specificity of this antibody is limited to soluble A-beta, sothat binding of aggregates or plaques cannot be expected.

A third anti-A-beta antibody, ponezumab (PF4360365), only binds toA-beta(x-40) molecules, and not to A-beta(x-42) molecules, the latterbeing assumed to be the (more) pathogenic A-beta species. Its affinityis even lower than the affinity of bapineuzumab, and the risk ofcerebral microhemorrhages can not yet be ruled out, due to its abilityto bind to A-beta plaques in blood vessels, combined with a remainingADCC/CDC activity of its Fc portion.

In summary, the above demonstrates that even if A-beta binding andclearance by (classical) antibodies appears to be an attractivemode-of-action for the development of therapeutical agents for thetreatment of e.g. AD, other characteristics and effects of suchimmunoglobulins which have not yet been fully elucidated, such as thepharmacological implications of their property to bind to certain formsof A-beta, make it far more difficult than one might have initiallyassumed to find and develop safe and efficient therapeutical antibodies.

Antibody fragments, such as immunoglobulin single variable domainantibodies or VHH domains (as defined below), having specificity forA-beta have also been described in the art: WO2004/44204; WO2006/40153;WO2007/35092; WO2008/122441; and WO2009/04494. Binding characteristicsof VHHs synthesized by the present inventors in accordance with theabove WO publications were unsatisfactory, for which reason they are notsupposed to enter clinical development.

WO09/149185 discloses so-called DVD constructs, and, inter alia, DVDconstructs having A-beta binding specificity. Upon combination of twodifferent anti-A-bata variable domains in such DVD constructs, bindingof the parental antibodies was maintained, but no increase in affinitywas observed by this combination. Moreover, the disclosed DVD constructscontain an Fc part which is present in “classical” antibodies, so thatside effects caused by Fc effector functions, such ascomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC), cannot be avoided (cf. above).

Definitive diagnosis of AD still requires post-mortem pathologicalexamination of the brain to demonstrate the presence of amyloid plaques,neurofibrillary tangles, synaptic loss and neuronal degeneration. Thisis essentially the same procedure as defined by Alois Alzheimer in 1906.In 1984 the National Institute of Neurological and CommunicativeDisorders and Stroke and the Alzheimer's Disease and Related DisordersAssociation (NINCDS-ADRDA) established formal criteria for the diagnosisof AD (reviewed in Petrella et al. (2003), Radiology 226, 315-336).Patients meeting all of the following criteria are diagnosed probableAD: dementia evidenced by examination and testing (e.g. Mini-MentalTest, Blessed Dementia Scale, or similar tests), impairment of memoryand at least one other cognitive function, normal consciousness, onsetbetween 40 and 90 years of age, absence of signs of other diseases thatcause dementia (exclusion criterion). A gradual progressive, cognitiveimpairment without an identifiable cause will be diagnosed as possibleAD. Probable AD is further defined as mild (early), moderate (middle) orsevere (late) dementia. Laboratory analysis is used to objectivelydefine or exclude alternative causes of dementia. ELISA assays ofA-beta(1-42) and phospho-tau in cerebrospinal fluid (CSF), combined withgenotyping for ApoE4 (a predisposing genetic factor) appear to besensitive and specific. The methods are, however, not widely applicablebecause of the invasive CSF puncture, preventing this to become routinescreening. ELISA for the neural thread protein (AD7C-NTP) (developed byNymox) demonstrated higher levels in urine from AD patients than fromnon-AD dementia patients or healthy controls. However, the mean levelswere significantly lower in early AD cases, suggesting the test is notreliable for testing for early onset of AD.

No biochemical method is as yet suited for the firm diagnosis of earlystages of AD, rather they merely help to confirm the clinical diagnosisof advanced cases. Clearly, more advanced techniques are needed to allowearly diagnosis before onset of clinical symptoms that signalirreversible brain damage.

Finally, not only for diagnostic purposes but also in e.g. pre-clinicalresearch and development, A-beta binding molecules are useful asresearch tools. Widely used are the antibodies already mentioned above,i.e. antibody 3D6 and antibody m266. Antibody 3D6 binds to A-beta with arelatively low affinity and may therefore not be suitable for allpurposes. Antibody m266 cross-reacts with N-terminally truncatedversions of A-beta, such as p3, which does not allow to distinguishbetween disease-relevant A-beta species, such as A-beta(1-40) andA-beta(1-42), and other molecules such as p3.

In view of the above, it is an object of the invention to providepharmacologically active agents, as well as compositions comprising thesame, that can be used in the prevention, treatment, alleviation and/ordiagnosis of diseases, disorders or conditions associated with A-betaand/or mediated by A-beta, such as AD, and to provide methods for theprevention, treatment, alleviation and/or diagnosis of such diseases,disorders or conditions, involving the use and/or administration of suchagents and compositions. Such agents may also be useful for doingresearch into the field of AD in general and, specifically, into theelucidation of AD disease mechanisms and potential therapeutic and/orprophylactic mechanisms.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods thatprovide certain advantages compared to the agents, compositions and/ormethods currently used and/or known in the art. These advantages includeimproved therapeutic and/or pharmacological properties and/or otheradvantageous properties (such as, for example, improved ease ofpreparation and/or reduced costs of goods), especially as compared toconventional antibodies against A-beta or fragments thereof as thosedescribed in the above section. Further advantages will become clearfrom the further description below.

More in particular, it is an object of the invention to provide novelA-beta binding molecules and, specifically, polypeptides binding tomammalian and, especially, human A-beta, wherein such molecules orpolypeptides are suitable for the above diagnostic, therapeutic andresearch purposes.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect of the invention, there are providedpolypeptides which comprise a first immunoglobulin single variabledomain which specifically binds to a first epitope of A-beta, and asecond immunoglobulin single variable domain which specifically binds toa second epitope of A-beta, wherein said first and said second epitopesof A-beta are not identical epitopes, i.e. are different epitopes, suchas e.g. the N-terminal epitope (SEQ ID NO:3) on the one hand and thecentral epitope (SEQ ID NO:4) on the other hand.

Preferably, said first and said second immunoglobulin single variabledomains each essentially consist of four framework regions (FR1 to FR4,respectively) and three complementarity determining regions (CDR1 to CDR3, respectively), wherein said first and said second immunoglobulinsingle variable domains are covalently linked by a linker peptide,wherein said linker peptide optionally comprises or consists of a thirdimmunoglobulin domain, such as e.g. a third immunoglobulin singlevariable domain.

Furthermore, said first and said second immunoglobulin single variabledomains are preferably antibody domains, more preferably VHH domains,and even more preferably humanized VHH domains.

The immunoglobulin single variable domains comprised in such polypeptideof the invention will typically have the structureFR(1)1-CDR(1)1-FR(1)2-CDR(1)2-FR(1)3-CDR(1)3-FR(1)4, andFR(2)1-CDR(2)1-FR(2)2-CDR(2)2-FR(2)3-CDR(2)3-FR(2)4, respectively.

Preferably, CDR(1)3 is selected from the group consisting of:

-   -   the amino acid sequences according to SEQ ID NO:13 and SEQ ID        NO:16; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequences according to SEQ ID NO:13 or SEQ ID        NO:16, respectively; and CDR(2)3 is selected from the group        consisting of:    -   the amino acid sequence according to SEQ ID NO:19; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequence according to SEQ ID NO:19.

Alternatively, CDR(1)3 is selected from the group consisting of:

-   -   the amino acid sequence according to SEQ ID NO:19; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequence according to SEQ ID NO:19; and

CDR(2)3 is selected from the group consisting of:

-   -   the amino acid sequences according to SEQ ID NO:13 and SEQ ID        NO:16; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequences according to SEQ ID NO:13 or SEQ ID        NO:16, respectively.

Especially useful polypeptides of the invention will include thefollowing CDR sequences (numbering as indicated in the paragraph above):

-   -   CDR(1)1: SEQ ID NO:11    -   CDR(1)2: SEQ ID NO:12    -   CDR(1)3: SEQ ID NO:13    -   CDR(2)1: SEQ ID NO:17    -   CDR(2)2: SEQ ID NO:18    -   CDR(2)3: SEQ ID NO:19        or:    -   CDR(1)1: SEQ ID NO:14    -   CDR(1)2: SEQ ID NO:15    -   CDR(1)3: SEQ ID NO:16    -   CDR(2)1: SEQ ID NO:17    -   CDR(2)2: SEQ ID NO:18    -   CDR(2)3: SEQ ID NO:19        or:    -   CDR(1)1: SEQ ID NO:17    -   CDR(1)2: SEQ ID NO:18    -   CDR(1)3: SEQ ID NO:19    -   CDR(2)1: SEQ ID NO:11    -   CDR(2)2: SEQ ID NO:12    -   CDR(2)3: SEQ ID NO:13        or:    -   CDR(1)1: SEQ ID NO:17    -   CDR(1)2: SEQ ID NO:18    -   CDR(1)3: SEQ ID NO:19    -   CDR(2)1: SEQ ID NO:14    -   CDR(2)2: SEQ ID NO:15    -   CDR(2)3: SEQ ID NO:16.

According to a specific embodiment of the invention, the polypeptides ofthe invention comprise, as a first immunoglobulin single variabledomain, the VHH domain ABII035 (SEQ ID NO:44), and as the secondimmunoglobulin single variable domain the VHH domain ABII059 (SEQ IDNO:45), or vice versa.

In an especially preferred embodiment, such polypeptide of the inventionadditionally comprises a half-life extending moiety, preferablycovalently linked to said polypeptide, such as an albumin binding moiety(e.g. an anti-albumin immunoglobulin domain), a transferrin bindingmoiety (e.g. an anti-transferrin immunoglobulin domain), a polyethyleneglycol molecule, a recombinant polyethylene glycol molecule, human serumalbumin, a fragment of human serum albumin, and an albumin bindingpeptide.

Very specific embodiments of the invention are the polypeptides havingan amino acid sequence as shown in SEQ ID NOs:26 to 31 (Fc fusionpolypeptides); SEQ ID NO:32 (HSA fusion polypeptide); SEQ ID NOs:34 to39 and 145 to 152 (albumin binding immunoglobulin single variable domainfusion polypeptides); and SEQ ID NOs:40 to 43, 142 and 143 (PEGylatedpolypeptides).

The polypeptides of the invention preferably bind, with one of itsimmunoglobulin single variable domains, to the A-beta epitope defined bySEQ ID NO:3 (N-terminal epitope), and with another immunoglobulin singlevariable domain to the A-beta epitope defined by SEQ ID NO:4 (centralepitope). Even more preferably, such polypeptides of the invention formcontacts to at least amino acids 1 (aspartate), 3 (glutamate), 19(phenylalanine), 20 (phenylalanine), and 23 (aspartate) of the humanA-beta peptide (SEQ ID NO:1). The IC50 values as measured in a TR-FRETbinding assay (using ABII002=SEQ ID NO:62 and ABII050=SEQ ID NO:100 ascompetitors; cf. Example 9.3) are preferably in the range of 10⁻⁹moles/liter or less, and more preferably in the range of from 5×10⁻¹⁹moles/liter to 10⁻¹² moles/liter.

According to another aspect, the invention relates to polypeptidescomprising or consisting of an immunoglobulin single variable domainessentially consisting of four framework regions (FR1 to FR4respectively) and three complementarity determining regions (CDR1 toCDR3 respectively), wherein said CDR sequences are defined as follows:

-   -   CDR1: SEQ ID NO:11    -   CDR2: SEQ ID NO:12    -   CDR3: SEQ ID NO:13        or    -   CDR1: SEQ ID NO:14    -   CDR2: SEQ ID NO:15    -   CDR3: SEQ ID NO:16        or    -   CDR1: SEQ ID NO:17    -   CDR2: SEQ ID NO:18    -   CDR3: SEQ ID NO:19,        and to polypeptides comprising or consisting of a VHH domain        having an amino acid sequence selected from the group consisting        of SEQ ID NOs: 47 to 111.

These polypeptides are useful building blocks or intermediates for theconstruction of biparatopic A-beta binding polypeptides in accordancewith the first aspect of the invention.

According to further aspects, the invention relates to nucleic acidmolecules, expression vectors, host cells, and methods of manufacturingused in the production of a polypeptide of the invention. Nucleic acidmolecules encoding the polypeptides of the invention can be used, in anisolated form, for constructing respective expression vectors, whichthen may be transfected into host cells used for biopharmaceuticalproduction of the polypeptides of the invention. Such method ofmanufacturing typically comprises the steps of culturing the host cellunder conditions that allow expression of the polypeptide, recoveringthe polypeptide and purifying it according to methods known in the art.

The polypeptides of the present invention are specifically useful inmethods of diagnosis, prevention, treatment and/or alleviation of thediseases, disorders and conditions as set out in detail below, and,especially, for the treatment of Alzheimer's disease (AD). Thus,according to this aspect, the polypeptides of the invention will be usedin the form of a pharmaceutical composition, i.e. as a medicament forthe treatment, alleviation or prevention of a disease, disorder orcondition, preferably in a human being, such disease, disorder orcondition being selected from the group consisting of neurodegenerativediseases or disorders, Alzheimer's disease, dementia of the Alzheimertype, cerebral amyloid angiopathy (CAA), trisomy 21 (Down's Syndrome),adult Down syndrome, hereditary cerebral hemorrhage with amyloidosis ofthe Dutch-type (HCHWA-D), dementia with Lewy Bodies, frontotemporallobar degeneration, glaucoma, amyotrophic lateral sclerosis, sporadicinclusion body myositis, and anxiety disorder in an elderly humansubject, or will be used for the diagnosis of such disease, disorder orcondition.

In addition to the above, the polypeptides of the present invention arespecifically useful for the treatment of dry AMD (age-related maculardegeneration) and glaucoma. The “dry” form of AMD, also known as“central geographic atrophy”, results from atrophy to the retinalpigment epithelial layer below the neurosensory retina, which causesvision loss through loss of photoreceptors (rods and cones) in thecentral part of the eye. No medical or surgical treatment is currentlyavailable for this condition. Treatments available so far (e.g.suggested by the National Eye Institute) include the use of vitaminsupplements with high doses of antioxidants, lutein and zeaxanthin,which may slow the progression of dry macular degeneration. Glaucoma isa disease where fluid pressure inside the eye increases, causingirreversible damage to the optic nerve and loss of vision. A-betacolocalizes with apoptotic retinal ganglion cells in experimentalglaucoma and induces significant retinal ganglion cell apoptosis in adose- and time-dependent manner.

For therapeutic purposes, the polypeptides of the invention orpharmaceutical compositions comprising such polypeptides may beadministered to a human being in need thereof by e.g. parenteral (esp.intravenous or subcutaneous) or intravitreal (esp. for the treatment ofdry AMD or glaucoma) injection.

Further aspects, embodiments and applications of the invention willbecome clear from the further description and the appended claimshereinbelow.

Unexpectedly, although intensive research into A-beta binding monoclonalantibodies has already been carried out and although highly specificantibodies with high affinity to A-beta are already available, theinventors succeeded to provide a group of A-beta binding molecules whichhave improved characteristics, e.g. are having still even better IC50values than e.g. antibodies 3D6 and m266 (cf. e.g. Table XVI), thehumanized counterparts of which are currently tested for therapeuticaluse in humans.

In addition, although it could be demonstrated that anti-A-betaantibodies known in the art are indeed able to reduce amyloid plaqueload in transgenic mice overproducing A-beta (maximal plaque loadreduction achieved by administration of monoclonal antibody m266 or itshumanized counterpart having been published to be about 60%), e.g.antibody m266 lacks amyloid plaque binding. Thus, such conventionalantibody may be expected to have less potential in situations where adirect binding of the anti-A-beta molecule to A-beta present in amyloidaggregates brings about additional benefit. In contrast thereto, theinventors were able to generate A-beta binding molecules which bind toamyloid plaques and are therefore expected to reduce or remove vascularamyloid by promoting physical dissociation, without increasing the riskor inducing microhemorrhages. As vascular amyloid is associated with theseverity of AD, this advantage of the polypeptides of the invention mayprove to be particularly useful in the treatment of later-stage orsevere AD, i.e. they can be expected to be particularly useful for thetreatment of patients which have a high brain amyloid plaque load,accumulated over many years.

Thus, in summary, the higher affinity of the A-beta binding polypeptidesof the invention and their unique plaque binding capabilities provide anunexpected superiority as compared to conventional anti-A-betaantibodies described in the art.

Another antibody currently in clinical trials, Ponezumab, has aspecificity for A-beta(x-40) and does not bind to A-beta(x-42), themajor pathogenic species in AD, and is therefore expected to shift theA-beta(x-40):A-beta(x-42) balance towards the toxic A-beta(x-42)species. In contrast thereto, the present inventors succeeded to provideA-beta binding molecules which, by binding to at least two differentepitopes, are able to capture the A-beta(x-40) and the A-beta(x-42) inorder to neutralize toxic effects of both species.

When compared to conventional antibodies in general, the polypeptides ofthe invention can be manufactured much easier, quicker and cheaper, havea higher stability and low antigenicity, and may be suitable for moreconvenient administration routes than injection or infusion, due totheir small size and structure. More specifically, production of thepolypeptides of the invention through fermentation in convenientrecombinant host organisms such as E. coli and yeast is cost-effective,as compared to the manufacture of conventional antibodies which requireexpensive mammalian cell culture facilities. Furthermore, achievablelevels of expression are high and yields of the polypeptides of theinvention are in the range of 1 to 10 g/l (E. coli) and up to 10 g/l(yeast) and more. The polypeptides of the invention are more soluble,meaning they may be stored and/or administered in higher concentrationscompared to conventional antibodies. They are stable at roomtemperature, meaning they may be prepared, stored and/or transportedwithout the use of refrigeration equipment, conveying cost, time andenvironmental savings. The polypeptides of the present invention alsoexhibit a prolonged stability at extremes of pH, meaning they would besuitable for delivery by oral administration.

Furthermore, the polypeptides of the invention do not need to comprisean Fc part which is present in “classical” antibodies, so that sideeffects caused by Fc effector functions, such as complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC),can be avoided. When used in anti-A-beta passive immunizations,conventional antibodies (having an Fc part) are suspected to account forthe induction of microhemorrhages observed in humans and animal models,which are associated with a targeting of cerebral vascular A-betadeposits (Cerebral amyloid angiopathy) leading to microbleedings viaADCC and/or CDC (Wilcock, D M, Colton, C A, CNS Neurol. Disord. DrugTargets (2009) Vol. 8(1):50-64).

Thus, in summary, the polypeptides of the invention combine theadvantageous characteristics of conventional antibodies, such as highspecificity and high selectivity, with the advantages as outlined above,and have surprisingly improved characteristics with regard to affinityand specificity as compared to conventional A-beta binding antibodies.

When compared to A-beta binding VHH domains already known in the art,such as the VHHs described in WO2006/040153, WO2007/35092 andWO2004/44204, the polypeptides of the invention show significantlyimproved binding characteristics, with respect to the binding tomonomeric A-beta as well as with respect to binding to aggregated A-beta(cf. e.g. Examples 3.2 and 6 below). Moreover, the polypeptidesaccording to the invention do bind to both monomeric A-beta as well asamyloid plaques, in contrast to VHH domains as described e.g. inWO2008/122441 or in Habicht et al. (2007), Proc. Natl. Acad. Sci. USA;104(49):19232-19237, which only bind to aggregated amyloid plaques.

Even more, the VHH domains of A-beta binding polypeptides of theinvention bind equally potent to human and rodent A-beta, as shown e.g.in Example 7 and FIG. 2. Binding of the biparatopic polypeptide of theinvention even increases the affinity to human as well as rodent A-betaby a factor of at least 103, as compared to the single VHH domains.Thus, the polypeptides of the invention are ideal detection tools acrossspecies for the disease-relevant A-beta forms, such as A-beta(1-40) andA-beta(1-42), allowing the use of esp. rodent animal models forpreclinical and scientific research. They provide superiority overantibody 3D6 which binds to A-beta with a significantly lower affinityand which is only weakly cross-reactive to rodent A-beta. There is alsosuperiority as compared to antibody m266 which recognizes rodent andhuman A-beta equally well, but cross-reacts with N-terminally truncatedversions of A-beta, such as p3 so that this antibody is not useful forcertain tests or assays relying on the specific detection ofdisease-relevant A-beta species, such as A-beta(1-40) and A-beta(1-42).

Finally, biparatopic anti-A-beta VHH constructs according to theinvention gain affinity by a factor of >1000 fold when two VHH detectingtwo different A-beta epitopes were combined, which is in clear contrastto those constructs disclosed in WO09/149,185.

Of course, the polypeptides of the invention are also useful fordiagnostic purposes, based on their affinity (sensitivity), specificity(with regard to epitopes as well as to species), and othercharacteristics thereof as outlined above, as compared to other A-betabinding molecules known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the decrease of free/unbound A-beta(1-40) in plasma afteri.p. administration of ABII320, ABII322, 3D6-IgG and m266-IgG, asdetected 2 hrs after injection, in APP transgenic mice (n=3). Depictedin the first column (unfilled box): vehicle (PBS); second column:ABII320 (132 nmol/kg); third column: ABII322 (132 nmol/kg); fourthcolumn: IgG 3D6 (132 nmol/kg); fifth column: IgG m266 (66.6 nmol/kg);concentrations for IgGs are calculated per binding site (2 binding sitesper IgG molecule).

FIG. 2 shows the binding of a biparatopic anti-A-beta VHH construct,including VHH domains ABII035 and ABII059, to human and rodent (mouse)A-beta

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The above and other aspects and embodiments of the invention will becomeclear from the further description herein, in which:

a) Unless indicated or defined otherwise, all terms used have theirusual meaning in the art, which will be clear to the skilled person.Reference is for example made to the standard handbooks, such asSambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd Ed.),Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); Lewin, “GenesIV”, Oxford University Press, New York, (1990), and Roitt et al.,“Immunology” (2^(nd) Ed.), Gower Medical Publishing, London, New York(1989), as well as to the general background art cited herein;Furthermore, unless indicated otherwise, all methods, steps, techniquesand manipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks, to the general background art referred to above andto the further references cited therein;b) Unless indicated otherwise, the terms “immunoglobulin” and“immunoglobulin sequence”—whether used herein to refer to a heavy chainantibody or to a conventional 4-chain antibody—are used as general termsto include both the full-size antibody, the individual chains thereof,as well as all parts, domains or fragments thereof (including but notlimited to antigen-binding domains or fragments such as VHH domains orVH/VL domains, respectively). In addition, the term “sequence” as usedherein (for example in terms like “immunoglobulin sequence”, “antibodysequence”, “(single) variable domain sequence”, “VHH sequence” or“protein sequence”), should generally be understood to include both therelevant amino acid sequence as well as nucleic acid sequences ornucleotide sequences encoding the same, unless the context requires amore limited interpretation;c) The term “domain” (of a polypeptide or protein) as used herein refersto a folded protein structure which has the ability to retain itstertiary structure independently of the rest of the protein. Generally,domains are responsible for discrete functional properties of proteins,and in many cases may be added, removed or transferred to other proteinswithout loss of function of the remainder of the protein and/or of thedomain.d) The term “immunoglobulin domain” as used herein refers to a globularregion of an antibody chain (such as e.g. a chain of a conventional4-chain antibody or of a heavy chain antibody), or to a polypeptide thatessentially consists of such a globular region. Immunoglobulin domainsare characterized in that they retain the immunoglobulin foldcharacteristic of antibody molecules, which consists of a 2-layersandwich of about 7 antiparallel beta-strands arranged in twobeta-sheets, optionally stabilized by a conserved disulphide bond.e) The term “immunoglobulin variable domain” as used herein means animmunoglobulin domain essentially consisting of four “framework regions”which are referred to in the art and hereinbelow as “framework region 1”or “FR1”; as “framework region 2” or“FR2”; as “framework region 3” or“FR3”; and as “framework region 4” or “FR4”, respectively; whichframework regions are interrupted by three “complementarity determiningregions” or “CDRs”, which are referred to in the art and hereinbelow as“complementarity determining region 1” or “CDR1”; as “complementaritydetermining region 2” or “CDR2”; and as “complementarity determiningregion 3” or “CDR3”, respectively. Thus, the general structure orsequence of an immunoglobulin variable domain can be indicated asfollows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulinvariable domain(s) that confer specificity to an antibody for theantigen by carrying the antigen-binding site.f) The term “immunoglobulin single variable domain” as used herein meansan immunoglobulin variable domain which is capable of specificallybinding to an epitope of the antigen without pairing with an additionalvariable immunoglobulin domain. One example of immunoglobulin singlevariable domains in the meaning of the present invention are “domainantibodies”, such as the immunoglobulin single variable domains VH andVL (VH domains and VL domains). Another example of immunoglobulin singlevariable domains are “VHH domains” (or simply “VHHs”) from camelids, asdefined hereinafter.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)2 fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an immunoglobulin singlevariable domain, as, in these cases, binding to the respective epitopeof an antigen would normally not occur by one (single) immunoglobulindomain but by a pair of (associating) immunoglobulin domains such aslight and heavy chain variable domains, i.e. by a VH-VL pair ofimmunoglobulin domains, which jointly bind to an epitope of therespective antigen.

f1) “VHH domains”, also known as VHHs, V_(H)H domains, VHH antibodyfragments, and VHH antibodies, have originally been described as theantigen binding immunoglobulin (variable) domain of “heavy chainantibodies” (i.e. of “antibodies devoid of light chains”;Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C,Songa E B, Bendahman N, Hamers R.: “Naturally occurring antibodiesdevoid of light chains”; Nature 363, 446-448 (1993)). The term “VHHdomain” has been chosen in order to distinguish these variable domainsfrom the heavy chain variable domains that are present in conventional4-chain antibodies (which are referred to herein as “V_(H) domains” or“VH domains”) and from the light chain variable domains that are presentin conventional 4-chain antibodies (which are referred to herein as“V_(L) domains” or “VL domains”). VHH domains can specifically bind toan epitope without an additional antigen binding domain (as opposed toVH or VL domains in a conventional 4-chain antibody, in which case theepitope is recognized by a VL domain together with a VH domain). VHHdomains are small, robust and efficient antigen recognition units formedby a single immunoglobulin domain.

In the context of the present invention, the terms VHH domain, VHH,V_(H)H domain, VHH antibody fragment, VHH antibody, as well as“Nanobody®” and “Nanobody® domain” (“Nanobody” being a trademark of thecompany Ablynx N.V.; Ghent; Belgium) are used interchangeably and arerepresentatives of immunoglobulin single variable domains (having thestructure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to anepitope without requiring the presence of a second immunoglobulinvariable domain), and which are distinguished from VH domains by theso-called “hallmark residues”, as defined in e.g. WO2009/109635, FIG. 1.

The amino acid residues of a VHH domain are numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to VHH domains fromCamelids, as shown e.g. in FIG. 2 of Riechmann and Muyldermans, J.Immunol. Methods 231, 25-38 (1999). According to this numbering,

-   -   FR1 comprises the amino acid residues at positions 1-30,    -   CDR1 comprises the amino acid residues at positions 31-35,    -   FR2 comprises the amino acids at positions 36-49,    -   CDR2 comprises the amino acid residues at positions 50-65,    -   FR3 comprises the amino acid residues at positions 66-94,    -   CDR3 comprises the amino acid residues at positions 95-102, and    -   FR4 comprises the amino acid residues at positions 103-113.

However, it should be noted that—as is well known in the art for V_(H)domains and for VHH domains—the total number of amino acid residues ineach of the CDRs may vary and may not correspond to the total number ofamino acid residues indicated by the Kabat numbering (that is, one ormore positions according to the Kabat numbering may not be occupied inthe actual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence.

Alternative methods for numbering the amino acid residues of V_(H)domains, which methods can also be applied in an analogous manner to VHHdomains, are known in the art. However, in the present description,claims and figures, the numbering according to Kabat and applied to VHHdomains as described above will be followed, unless indicated otherwise.

The total number of amino acid residues in a VHH domain will usually bein the range of from 110 to 120, often between 112 and 115. It shouldhowever be noted that smaller and longer sequences may also be suitablefor the purposes described herein.

Further structural characteristics and functional properties of VHHdomains and polypeptides containing the same can be summarized asfollows:

VHH domains (which have been “designed” by nature to functionally bindto an antigen without the presence of, and without any interaction with,a light chain variable domain) can function as a single, relativelysmall, functional antigen-binding structural unit, domain orpolypeptide. This distinguishes the VHH domains from the VH and VLdomains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or immunoglobulin single variable domains, but need to becombined in some form or another to provide a functional antigen-bindingunit (as in for example conventional antibody fragments such as Fabfragments; in scFv's, which consist of a VH domain covalently linked toa VL domain).

Because of these unique properties, the use of VHH domains—either aloneor as part of a larger polypeptide—offers a number of significantadvantages over the use of conventional VH and VL domains, scFv's orconventional antibody fragments (such as Fab- or F(ab′)2-fragments):

-   -   only a single domain is required to bind an antigen with high        affinity and with high selectivity, so that there is no need to        have two separate domains present, nor to assure that these two        domains are present in the right spacial conformation and        configuration (i.e. through the use of especially designed        linkers, as with scFv's);    -   VHH domains can be expressed from a single gene and require no        post-translational folding or modifications;    -   VHH domains can easily be engineered into multivalent and        multispecific formats (as further discussed herein);    -   VHH domains are highly soluble and do not have a tendency to        aggregate (as with the mouse-derived antigen-binding domains        described by Ward et al., Nature 341: 544-546 (1989));    -   VHH domains are highly stable to heat, pH, proteases and other        denaturing agents or conditions and, thus, may be prepared,        stored or transported without the use of refrigeration        equipments, conveying a cost, time and environmental savings;    -   VHH domains are easy and relatively cheap to prepare, even on a        scale required for production. For example, VHH domains and        polypeptides containing the same can be produced using microbial        fermentation (e.g. as further described below) and do not        require the use of mammalian expression systems, as with for        example conventional antibody fragments;    -   VHH domains are relatively small (approximately 15 kDa, or 10        times smaller than a conventional IgG) compared to conventional        4-chain antibodies and antigen-binding fragments thereof, and        therefore        -   show high(er) penetration into tissues and        -   can be administered in higher doses            than such conventional 4-chain antibodies and            antigen-binding fragments thereof;    -   VHH domains can show so-called cavity-binding properties (inter        alia due to their extended CDR3 loop, compared to conventional        VH domains) and can therefore also access targets and epitopes        not accessible to conventional 4-chain antibodies and        antigen-binding fragments thereof.

Methods of obtaining VHH domains binding to a specific antigen orepitope have been described earlier, e.g. in WO2006/040153 andWO2006/122786. As also described therein in detail, VHH domains derivedfrom camelids can be “humanized” by replacing one or more amino acidresidues in the amino acid sequence of the original VHH sequence by oneor more of the amino acid residues that occur at the correspondingposition(s) in a VH domain from a conventional 4-chain antibody from ahuman being. A humanized VHH domain can contain one or more fully humanframework region sequences, and, in an even more specific embodiment,can contain human framework region sequences derived from DP-29, DP-47,DP-51, or parts thereof, optionally combined with JH sequences, such asJH5.

f2) “Domain antibodies”, also known as “Dab”s, “Domain Antibodies”, and“dAbs” (the terms “Domain Antibodies” and “dAbs” being used astrademarks by the GlaxoSmithKline group of companies) have beendescribed in e.g. Ward, E. S., et al.: “Binding activities of arepertoire of single immunoglobulin variable domains secreted fromEscherichia coli”; Nature 341: 544-546 (1989); Holt, L. J. et al.:“Domain antibodies: proteins for therapy”; TRENDS in Biotechnology21(11): 484-490 (2003); and WO2003/002609.

Domain antibodies essentially correspond to the VH or VL domains ofnon-camelid mammalians, in particular human 4-chain antibodies. In orderto bind an epitope as a single antigen binding domain, i.e. withoutbeing paired with a VL or VH domain, respectively, specific selectionfor such antigen binding properties is required, e.g. by using librariesof human single VH or VL domain sequences. Domain antibodies have, likeVHHs, a molecular weight of approximately 13 to approximately 16 kDaand, if derived from fully human sequences, do not require humanizationfor e.g. therapeutical use in humans. As in the case of VHH domains,they are well expressed also in prokaryotic expression systems,providing a significant reduction in overall manufacturing cost.

Domain antibodies, as well as VHH domains, can be subjected to affinitymaturation by introducing one or more alterations in the amino acidsequence of one or more CDRs, which alterations result in an improvedaffinity of the resulting immunoglobulin single variable domain for itsrespective antigen, as compared to the respective parent molecule.Affinity-matured immunoglobulin single variable domain molecules of theinvention may be prepared by methods known in the art, for example, asdescribed by Marks et al., 1992, Biotechnology 10:779-783, or Barbas, etal., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813.; Shier et al., 1995,Gene 169:147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jacksonet al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J.Mol. Biol. 226(3): 889 896; KS Johnson and RE Hawkins, “Affinitymaturation of antibodies using phage display”, Oxford University Press1996.

f3) Furthermore, it will also be clear to the skilled person that it ispossible to “graft” one or more of the CDR's mentioned above onto other“scaffolds”, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting are known in the art.g) The terms “epitope” and “antigenic determinant”, which can be usedinterchangeably, refer to the part of a macromolecule, such as apolypeptide, that is recognized by antigen-binding molecules, such asconventional antibodies or the polypeptides of the invention, and moreparticularly by the antigen-binding site of said molecules. Epitopesdefine the minimum binding site for an immunoglobulin, and thusrepresent the target of specificity of an immunoglobulin.

The part of an antigen-binding molecule (such as a conventional antibodyor a polypeptide of the invention) that recognizes the epitope is calleda paratope.

h) The term “biparatopic” (antigen-)binding molecule or “biparatopic”polypeptide as used herein shall mean a polypeptide comprising a firstimmunoglobulin single variable domain and a second immunoglobulin singlevariable domain as herein defined, wherein these two variable domainsare capable of binding to two different epitopes of one antigen, whichepitopes are not normally bound at the same time by one monospecificimmunoglobulin, such as e.g. a conventional antibody or oneimmunoglobulin single variable domain. The biparatopic polypeptidesaccording to the invention are composed of variable domains which havedifferent epitope specificities, and do not contain mutuallycomplementary variable domain pairs which bind to the same epitope. Theydo therefore not compete with each other for binding to A-beta.i) A polypeptide (such as an immunoglobulin, an antibody, animmunoglobulin single variable domain, a polypeptide of the invention,or generally an antigen binding molecule or a fragment thereof) that can“bind to” or “specifically bind to”, that “has affinity for” and/or that“has specificity for” a certain epitope, antigen or protein (or for atleast one part, fragment or epitope thereof) is said to be “against” or“directed against” said epitope, antigen or protein or is a “binding”molecule with respect to such epitope, antigen or protein.k) Generally, the term “specificity” refers to the number of differenttypes of antigens or epitopes to which a particular antigen-bindingmolecule or antigen-binding protein (such as an immunoglobulin, anantibody, an immunoglobulin single variable domain, or a polypeptide ofthe invention) can bind. The specificity of an antigen-binding proteincan be determined based on its affinity and/or avidity. The affinity,represented by the equilibrium constant for the dissociation of anantigen with an antigen-binding protein (KD), is a measure for thebinding strength between an epitope and an antigen-binding site on theantigen-binding protein: the lesser the value of the KD, the strongerthe binding strength between an epitope and the antigen-binding molecule(alternatively, the affinity can also be expressed as the affinityconstant (KA), which is 1/KD). As will be clear to the skilled person(for example on the basis of the further disclosure herein), affinitycan be determined in a manner known per se, depending on the specificantigen of interest. Avidity is the measure of the strength of bindingbetween an antigen-binding molecule (such as an immunoglobulin, anantibody, an immunoglobulin single variable domain, or a polypeptide ofthe invention) and the pertinent antigen. Avidity is related to both theaffinity between an epitope and its antigen binding site on theantigen-binding molecule and the number of pertinent binding sitespresent on the antigen-binding molecule.

Typically, antigen-binding proteins (such as the polypeptides of theinvention) will bind with a dissociation constant (K_(D)) of 10E-5 to10E-14 moles/liter (M) or less, and preferably 10E-7 to 10E-14moles/liter (M) or less, more preferably 10E-8 to 10E-14 moles/liter,and even more preferably 10E-11 to 10E-13 (as measured e.g. in a Kinexaassay as described in Example 9.7), and/or with an association constant(K_(A)) of at least 10E7 ME-1, preferably at least 10E8 ME-1, morepreferably at least 10E9 ME-1, such as at least 10E11 ME-1. Any K_(D)value greater than 10E-4 M is generally considered to indicatenon-specific binding. Preferably, a polypeptide of the invention willbind to the desired antigen with a K_(D) less than 500 nM, preferablyless than 200 nM, more preferably less than 10 nM, such as less than 500pM. Specific binding of an antigen-binding protein to an antigen orepitope can be determined in any suitable manner known per se,including, for example, the assays described herein, Scatchard analysisand/or competitive binding assays, such as radioimmunoassays (RIA),enzyme immunoassays (EIA) and sandwich competition assays, and thedifferent variants thereof known per se in the art.

l) Amino acid residues will be indicated according to the standardthree-letter or one-letter amino acid code, as generally known andagreed upon in the art. When comparing two amino acid sequences, theterm “amino acid difference” refers to insertions, deletions orsubstitutions of the indicated number of amino acid residues at aposition of the reference sequence, compared to a second sequence. Incase of substitution(s), such substitution(s) will preferably beconservative amino acid substitution(s), which means that an amino acidresidue is replaced with another amino acid residue of similar chemicalstructure and which has little or essentially no influence on thefunction, activity or other biological properties of the polypeptide.Such conservative amino acid substitutions are well known in the art,for example from WO 98/49185, wherein conservative amino acidsubstitutions preferably are substitutions in which one amino acidwithin the following groups (i)-(v) is substituted by another amino acidresidue within the same group: (i) small aliphatic, nonpolar or slightlypolar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negativelycharged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;(iii) polar, positively charged residues: His, Arg and Lys; (iv) largealiphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v)aromatic residues: Phe, Tyr and Trp. Particularly preferred conservativeamino acid substitutions are as follows:Ala into Gly or into Ser;Arg into Lys;Asn into Gln or into His;Asp into Glu;Cys into Ser;Gln into Asn;Glu into Asp;Gly into Ala or into Pro;His into Asn or into Gln;Ile into Leu or into Val;Leu into Ile or into Val;Lys into Arg, into Gln or into Glu;Met into Leu, into Tyr or into Ile;Phe into Met, into Leu or into Tyr;Ser into Thr;Thr into Ser;Trp into Tyr;Tyr into Trp or into Phe;Val into Ile or into Leu.m) A nucleic acid or polypeptide molecule is considered to be “(in)essentially isolated (form)”—for example, when compared to its nativebiological source and/or the reaction medium or cultivation medium fromwhich it has been obtained—when it has been separated from at least oneother component with which it is usually associated in said source ormedium, such as another nucleic acid, another protein/polypeptide,another biological component or macromolecule or at least onecontaminant, impurity or minor component. In particular, a nucleic acidor polypeptide molecule is considered “essentially isolated” when it hasbeen purified at least 2-fold, in particular at least 10-fold, more inparticular at least 100-fold, and up to 1000-fold or more. A nucleicacid or polypeptide molecule that is “in essentially isolated form” ispreferably essentially homogeneous, as determined using a suitabletechnique, such as a suitable chromatographical technique, such aspolyacrylamide-gelelectrophoresis;n) “Sequence identity” between e.g. two immunoglobulin single variabledomain sequences indicates the percentage of amino acids that areidentical between these two sequences. It may be calculated ordetermined as described in paragraph f) on pages 49 and 50 ofWO08/020,079. “Sequence similarity” indicates the percentage of aminoacids that either are identical or that represent conservative aminoacid substitutions.Target Specificity

The polypeptides of the invention have specificity for A-beta in thatthey comprise immunoglobulin single variable domains specificallybinding to one or more A-beta molecules and, more precisely, to epitopeswithin the A-beta molecule(s). A-beta may adopt and may occur e.g. inthe human body in different forms, such as in monomeric form, in oligo-and multimeric forms, in aggregated soluble and insoluble forms, infibrillar form, in proto-fibrillar form, in the form of amyloid plaquesand deposits that are present in the central nervous system, skeletalmuscle, platelets, vascular system, pancreas, kidney, spleen, heart,liver, testis, aorta, lung, intestine, skin, adrenal, salivary, andthyroid glands (Roher et al. Alzheimer's & Dementia 5 (2009) p. 18-29).It is within the scope of the invention that the polypeptides of theinvention bind to any of the forms in which A-beta may occur, andespecially to the forms that are most relevant from a biological and/ortherapeutic point of view.

The polypeptides of the invention may bind to A-beta peptide moleculesof different length, such as A-beta(1-42) which consists of the aminoacid sequence shown as SEQ ID NO:1, A-beta(1-40) which consists of theamino acids 1 to 40 of the amino acid sequence shown as SEQ ID NO:1,A-beta(1-39) (amino acids 1 to 39), A-beta(1-38) (amino acids 1 to 38),A-beta(1-37) (amino acids 1 to 37), and the like. Binding of thepolypeptide of the invention may occur at the N-terminal end, theC-terminal end, or somewhere in between.

Also, the invention is not limited with regard to the species form ofA-beta. Thus, the polypeptides of the invention may preferably bind tohuman A-beta (SEQ ID NO:1), if intended for therapeutic purposes.However, polypeptides binding to e.g. other warm-blooded animal or,preferably, mammalian forms of A-beta are within the scope of theinvention as well. A polypeptide of the invention binding to one speciesform of A-beta may cross-react with A-beta from one or more otherspecies. For example, polypeptides of the invention binding to humanA-beta may or may not show cross-reactivity with A-beta from one or moreother species of primates and/or with A-beta from one or more species ofanimals that are often used in animal models for diseases (for examplemouse—cf. SEQ ID NO:2, rat, rabbit, pig or dog), and in particular inanimal models for diseases and disorders associated with A-beta (such asthe species and animal models mentioned herein). Polypeptides of theinvention that show such cross-reactivity may have advantages from aresearch and/or drug development point of view, since it allows thepolypeptides of the invention binding to human A-beta to be tested inimportant disease models such as mice or rats.

Also, the invention is not limited to or defined by a specific antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) of A-beta against which the polypeptides of the inventionare directed. Some of the preferred epitopes of A-beta against which thepolypeptides of the present invention may be directed are the epitopesused for immunotherapy, and in particular for passive immunotherapy ofAD. For example, as mentioned in Weksler M., Immunity and Ageing 1, 2(2004) and in the background art referred to therein, it is known thatthere are three major epitopes on A-beta, i.e. an N-terminal epitope(amino acids 1-16 of A-beta: DAEFRHDSGYEVHHQK; SEQ ID NO:3), a centralepitope (amino acids 16-28: KLVFFAEDVGSNK; SEQ ID NO:4) and a C-terminalepitope (amino acids 28-42; KGAIIGLMVGGVVIA; SEQ ID NO:5). Thepolypeptides of the invention may be directed against either of theseepitopes. Specifically preferred are polypeptides of the inventioncomprising at least two immunoglobulin single variable domains, whereinone immunoglobulin single variable domain binds to the N-terminalepitope and a second immunoglobulin single variable domain binds to thecentral epitope.

Polypeptides of the Invention

In its broadest sense, the invention provides novel pharmaceuticallyactive agents for the prevention, treatment, alleviation and/ordiagnosis of A-beta associated diseases, disorders or conditions and,specifically, AD. The agents according to the invention belong to anovel class of A-beta binding molecules, namely biparatopic polypeptidescomprising two or more immunoglobulin single variable domains binding tothe antigen A-beta at different epitopes. More specifically, suchpolypeptide of the invention essentially consists of or comprises (i) afirst immunoglobulin single variable domain specifically binding to afirst epitope of A-beta and (ii) a second immunoglobulin single variabledomain specifically binding to a second epitope of A-beta, wherein thefirst epitope of A-beta and the second epitope of A-beta are notidentical epitopes. In other words, such polypeptide of the inventioncomprises or essentially consist of two or more immunoglobulin singlevariable domains that are directed against at least two differentepitopes present in A-beta, wherein said immunoglobulin single variabledomains are linked to each other in such a way that they are capable ofsimultaneously binding A-beta. In this sense, the polypeptide of theinvention can also be regarded as a “multivalent” immunoglobulinconstruct, and especially as a “multivalent immunoglobulin singlevariable domain construct”, in that the whole polypeptide includes atleast two binding sites for A-beta.

A polypeptide of the invention includes (at least) two anti-A-betaimmunoglobulin single variable domains, wherein (the) two immunoglobulinsingle variable domains are directed against different epitopes withinthe A-beta molecule. Thus, these two immunoglobulin single variabledomains will have a different epitope specificity and thereforedifferent CDR sequences. For this reason, polypeptides of the inventionwill herein also be named “biparatopic polypeptides”, or “biparatopicdomain antibody constructs” (if the immunoglobulin single variabledomains consist or essentially consist of domain antibodies), or“biparatopic Nanobody constructs” or “biparatopic VHH domainconstructs”, or “biparatopic VHH constructs” (if the immunoglobulinsingle variable domains consist or essentially consist of Nanobodies orVHH domains), respectively, as the two immunoglobulin single variabledomains will include two different paratopes.

According to a specific embodiment of the invention, in case that thepolypeptide of the invention includes more than two anti-A-betaimmunoglobulin single variable domains, i.e. three, four or even moreanti-A-beta immunoglobulin single variable domains, at least two of theanti-A-beta immunoglobulin single variable domains are directed againstdifferent epitopes within the A-beta molecule, wherein any furtherimmunoglobulin single variable domain may bind to any of these twodifferent epitopes and/or a further epitope present in the A-betamolecule.

According to another specific embodiment of the invention, thepolypeptide of the invention can, in addition to the two anti-A-betaimmunoglobulin single variable domains described above, include anyother additional moiety, such as a linker (as described in more detailbelow) and/or additional protein domains, such as e.g. a furtherimmunoglobulin single variable domain (as described in more detailbelow), as long as its binding to A-beta will not be prevented by suchadditional moiety. The polypeptide of the invention can additionallycontain modifications such as glycosyl residues, modified amino acidside chains, and the like.

As set out before, two immunoglobulin single variable domains within onepolypeptide of the invention will bind to different epitopes of A-beta.This can be achieved in one of the following manners: Either, the twoimmunoglobulin single variable domains will bind the two epitopes withinone and the same A-beta molecule (intramolecular binding).Alternatively, they may bind epitopes located within two distinct A-betamolecules, i.e. one immunoglobulin single variable domain will bind toone epitope on one A-beta molecule, whereas the other immunoglobulinsingle variable domain will bind to the other epitope on another A-betamolecule, thereby cross-linking two A-beta molecules (intermolecularbinding).

According to a preferred embodiment, the polypeptide of the inventionwill bind the two epitopes within one and the same A-beta molecule, sothat no cross-linking will occur, and thepolypeptide-of-the-invention—A-beta complexes will form in astoichiometry of 1:1. Thus, (predominantly) intramolecular binding ispreferred as compared to (predominantly) intermolecular binding, itbeing understood that a minor fraction of intermolecular binding maynevertheless occur. A distinction between intra- and intermolecularbinding can be made using Biacore or size exclusion chromatorgraphyassays (as described by Santora et al., Anal. Biochem., 299:119-129)—cf. Example 8.2 hereinbelow. However, it should be noted thatpolypeptides that operate via intermolecular binding of separate A-betamolecules are also within the scope of this invention.

In another preferred embodiment of the invention, the first and thesecond anti-A-beta immunoglobulin single variable domains comprised in apolypeptide of the invention each essentially consists of 4 frameworkregions (FR1 to FR4 respectively) and 3 complementarity determiningregions (CDR1 to CDR3 respectively). Within the polypeptide of theinvention, said first and second immunoglobulin single variable domainsare covalently linked, optionally by a linker peptide as describedbelow, wherein such linker sterically allows for optimal binding of theat least two immunoglobulin single variable domains to the respectiveA-beta epitopes.

It will be clear to the skilled person that for pharmaceutical uses inhumans, the polypeptides of the invention are preferably directedagainst human A-beta, whereas for veterinary purposes, the polypeptidesof the invention are preferably directed against A-beta from the speciesto be treated.

It will also be clear to the skilled person that when used as atherapeutic agent in humans, the immunoglobulin single variable domainscomprised in the polypeptides according to the invention are preferablyhumanized immunoglobulin single variable domains.

According to the invention, the two or more immunoglobulin singlevariable domains can be, independently of each other: Domain antibodies,i.e. VL or VH antibody domains as described above, and/or VHH domains asdescribed above, and/or any other sort of immunoglobulin single variabledomains, provided that these immunoglobulin single variable domains willbind the antigen, i.e. A-beta, not by forming mutually complementaryvariable domain pairs jointly binding to the same epitope (as in thecase of e.g. a VL-VH domain pair of a conventional antibody), but byindependently binding to different epitopes (i.e. as a “biparatopic”antigen binding molecule as defined above).

According to a preferred embodiment of the invention, the first and thesecond immunoglobulin single variable domains essentially consist ofeither domain antibody sequences or VHH domain sequences as describedabove. According to a particularly preferred embodiment, the first andthe second immunoglobulin single variable domains essentially consist ofVHH domain sequences. Accordingly, the invention will herein and,especially, in the experimental part, be described in more detail withreference to biparatopic polypeptides comprising two (optionallyhumanized) anti-A-beta VHH domain sequences (VHHs) binding to twodifferent epitopes of A-beta, i.e. biparatopic VHH domain constructs.However, it will be clear to the skilled person that the teaching hereinmay be applied analogously to polypeptides including other anti-A-betaimmunoglobulin single variable domains, such as domain antibodies.

The polypeptides of the invention not only possess the advantageouscharacteristics of conventional antibodies, such as low toxicity andhigh selectivity, but they also exhibit additional properties. They aremore soluble, meaning they may be stored and/or administered in higherconcentrations compared to conventional antibodies. They are stable atroom temperature, meaning they may be prepared, stored and/ortransported without the use of refrigeration equipment, conveying cost,time and environmental savings. The polypeptides of the presentinvention also exhibit a prolonged stability at extremes of pH, meaningthey would be suitable for delivery by oral administration.

Furthermore, the polypeptides of the invention do not need to comprisean Fc part which is present in “classical” antibodies, so that sideeffects caused by Fc effector functions, such as complement-dependentcytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC),can be avoided. When used in anti-A-beta passive immunizations,conventional antibodies (having an Fc part) are suspected to account forthe induction of microhemorrhages observed in humans and animal models,which are associated with a targeting of cerebral vascular A-betadeposits (Cerebral amyloid angiopathy) leading to microbleedings viaADCC and/or CDC (Wilcock, D M, Colton, C A, CNS Neurol. Disord. DrugTargets (2009) Vol. 8(1):50-64).

According to another embodiment of the invention, the at least twoimmunoglobulin single variable domains present in a polypeptide of theinvention can be linked to each other directly (i.e. without use of alinker) or via a linker. The linker is preferably a linker peptide andwill, according to the invention, be selected so as to allow binding ofthe at least two different immunoglobulin single variable domains toeach of their at least two different epitopes of A-beta, either withinone and the same A-beta molecule, or within two different molecules.

Suitable linkers will inter alia depend on the epitopes and,specifically, the distance between the epitopes on A-beta to which theimmunoglobulin single variable domains bind, and will be clear to theskilled person based on the disclosure herein, optionally after somelimited degree of routine experimentation.

Also, when the two or more immunoglobulin single variable domains thatbind to A-beta are domain antibodies or VHH domains, they may also belinked to each other via a third domain antibody or VHH domain (in whichthe two or more immunoglobulin single variable domains may be linkeddirectly to the third domain antibody or VHH domain or via suitablelinkers). Such a third domain antibody or VHH domain may for example bea domain antibody or VHH domain that provides for an increasedhalf-life, as further described herein. For example, the latter domainantibody or VHH domain may be a domain antibody or VHH domain that iscapable of binding to a (human) serum protein such as (human) serumalbumin or (human) transferrin, as further described herein.

Alternatively, the two or more immunoglobulin single variable domainsthat bind to A-beta may be linked in series (either directly or via asuitable linker) and the third (single) domain antibody or VHH domain(which may provide for increased half-life, as described above) may beconnected directly or via a linker to one of these two or moreaforementioned immunoglobulin sequences.

Suitable linkers are described herein in connection with specificpolypeptides of the invention and may—for example and withoutlimitation—comprise an amino acid sequence, which amino acid sequencepreferably has a length of 9 or more amino acids, more preferably atleast 17 amino acids, such as about 20 to 40 amino acids. However, theupper limit is not critical but is chosen for reasons of convenienceregarding e.g. biopharmaceutical production of such polypeptides.

The linker sequence may be a naturally occurring sequence or anon-naturally occurring sequence. If used for therapeutical purposes,the linker is preferably non-immunogenic in the subject to which theanti-A-beta polypeptide of the invention is administered.

One useful group of linker sequences are linkers derived from the hingeregion of heavy chain antibodies as described in WO 96/34103 and WO94/04678.

Other examples are poly-alanine linker sequences such as Ala-Ala-Ala.

Further preferred examples of linker sequences are Gly/Ser linkers ofdifferent length such as (gly_(x)ser_(y))_(z) linkers, including(gly₄ser)₃, (gly₄ser)₄, (gly₄ser), (gly₃ser), gly₃, and (gly₃ser₂)₃.

If the polypeptide of the invention is modified by the attachment of apolymer, for example of a polyethylene glycol (PEG) moiety, the linkersequence preferably includes an amino acid residue, such as a cysteineor a lysine, allowing such modification, e.g. PEGylation, in the linkerregion. Preferred examples of such linkers are:

(“GS9, C5”, SEQ ID NO: 6) GGGGCGGGS (“GS25, C5, SEQ ID NO: 7)GGGGCGGGGSGGGGSGGGGSGGGGS (“GS27, C14”, SEQ ID NO: 8)GGGSGGGGSGGGGCGGGGSGGGGSGGG, (“GS35, C15”, SEQ ID NO: 9)GGGGSGGGGSGGGGCGGGGSGGGGSGGGGSGGGGS, and (“GS35, C5”, SEQ ID NO: 10)GGGGCGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS.

Some non-limiting examples of PEGylated polypeptides of the inventionincluding such linkers are shown in SEQ ID NOs 40 to 43, 142, and 143.

Furthermore, the linker may also be a poly(ethylene glycol) moiety, asshown in e.g. WO04/081026.

In another embodiment, the at least two immunoglobulin single variabledomains of the polypeptide of the invention are linked to each other viaanother moiety (optionally via one or two linkers), such as anotherpolypeptide which, in a preferred but non-limiting embodiment, may be afurther immunoglobulin single variable domain as already describedabove. Such moiety may either be essentially inactive or may have abiological effect such as improving the desired properties of thepolypeptide or may confer one or more additional desired properties tothe polypeptide. For example, and without limitation, the moiety mayimprove the half-life of the protein or polypeptide, and/or may reduceits immunogenicity or improve any other desired property.

Some non-limiting examples of such constructs are the constructs of SEQID NOs:34 to 39.

According to a preferred embodiment, the polypeptides of the inventioncomprise a first immunoglobulin single variable domain which binds tothe epitope as defined by SEQ ID NO:3, and a second immunoglobulinsingle variable domain which binds to the epitope as defined by SEQ IDNO:4, or a first immunoglobulin single variable domain which binds tothe epitope as defined by SEQ ID NO:4, and a second immunoglobulinsingle variable domain which binds to the epitope as defined by SEQ IDNO:3.

Even more preferred, contact between the immunoglobulin single variabledomains and the A-beta molecule is made as described in Examples 8.2 and8.3, i.e. the polypeptide of the invention forms contacts to at leastamino acids 1, 3, 13, 20, and 23 of the human or mouse A-beta peptide.

Preferably, the polypeptides of the invention are having dissociationconstant (K_(D)) values, measured in Kinexa assays as described inExample 9.7, in the range of 10⁻⁶ moles/liter or less, more preferably10⁻⁹ moles/liter or less, and even more preferably in the range of from10⁻¹¹ to 10⁻¹³ moles/liter, or are having an IC50 value as measured in aTR-FRET binding assay, as set out in Example 9.3, of 10⁻⁹ moles/liter orbelow, and preferably in the range of from 5×10⁻¹⁰ moles/liter to 10⁻¹²moles/liter.

According to an even more preferred embodiment of the invention, the CDRsequences in the polypeptide of the invention are as defined below andare also such that the polypeptide of the invention binds to A-beta witha dissociation constant (K_(D)) as set out in the paragraph above or arehaving IC50 values as set out above.

According to a specific embodiment of the invention, the polypeptide ofthe invention comprises two A-beta binding immunoglobulin singlevariable domains having the structure (SEQ ID NOs as given in Table Ibelow): Immunoglobulin single variable domain 1:

FR(1)1-CDR(1)1-FR(1)2-CDR(1)2-FR(1)3-CDR(1)3-FR(1)4, immunoglobulinsingle variable domain 2:

FR(2)1-CDR(2)1-FR(2)2-CDR(2)2-FR(2)3-CDR(2)3-FR(2)4,

wherein:

CDR(1)3 is selected from the group consisting of:

-   -   the amino acid sequences according to SEQ ID NO:13 and SEQ ID        NO:16; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequences according to SEQ ID NO:13 or SEQ ID        NO:16, respectively;        and        CDR(2)3 is selected from the group consisting of:    -   the amino acid sequence according to SEQ ID NO:19; and    -   amino acid sequences which have up to three, preferably up to        two, and more preferably one amino acid difference as compared        to said amino acid sequence according to SEQ ID NO:19;        and        wherein the other CDR sequences and the framework region        sequences are not specifically limited but will be selected by        the skilled person according to the specific needs, such as        humanized framework regions in case of a polypeptide intended        for use in humans in order to reduce immunogenicity of the        polypeptide of the invention. The order of the immunoglobulin        single variable domains 1 and 2 is not particularly limited, so        that, within a polypeptide of the invention, immunoglobulin        single variable domain 1 may be located N-terminally and        immunoglobulin single variable domain 2 may be located        C-terminally, or vice versa.

Preferably, CDR(1)3 is selected from the group consisting of amino acidsequences according to SEQ ID NO:13 and SEQ ID NO:16, and CDR(2)3 is theamino acid sequence according to SEQ ID NO:19.

Even more preferably, the polypeptide of the invention comprises twoA-beta binding immunoglobulin single variable domains having thestructure (SEQ ID NOs as given in Table I below):

Immunoglobulin single variable domain 1:

FR(1)1-CDR(1)1-FR(1)2-CDR(1)2-FR(1)3-CDR(1)3-FR(1)4, immunoglobulinsingle variable domain 2:

FR(2)1-CDR(2)1-FR(2)2-CDR(2)2-FR(2)3-CDR(2)3-FR(2)4,

wherein:

CDR(1)1 is the amino acid sequence according to SEQ ID NO:11 (which isthe same as the amino acid sequence according to SEQ ID NO:14);

CDR(1)2 is selected from the group consisting of amino acid sequencesaccording to SEQ ID NO:12 and SEQ ID NO:15;

CDR(1)3 is selected from the group consisting of amino acid sequencesaccording to SEQ ID NO:13 and SEQ ID NO:16;

CDR(2)1 is the amino acid sequence according to SEQ ID NO:17;

CDR(2)2 is the amino acid sequence according to SEQ ID NO:18; and

CDR(2)3 is the amino acid sequence according to SEQ ID NO:19;

and

wherein the framework region sequences are not specifically limited butwill be selected by the skilled person according to the specific needs,such as humanized framework regions in case of a polypeptide intendedfor use in humans in order to reduce immunogenicity of the polypeptideof the invention.

In the polypeptide of the invention as described above, the particularorder of the immunoglobulin single variable domains 1 and 2 as set outabove within the polypeptide is not critical, so that above-mentionedimmunoglobulin single variable domain 1 may be located at the N-terminalend of the polypeptide, followed by above-mentioned immunoglobulinsingle variable domain 2; alternatively, above-mentioned immunoglobulinsingle variable domain 2 may be located at the N-terminal end of thepolypeptide, followed by above-mentioned immunoglobulin single variabledomain 1. In both cases, additional sequences and moieties may bepresent within the polypeptide of the invention, e.g. N-terminally,C-terminally, or located between the two immunoglobulin single variabledomains, as set out in more detail herein.

The above CDR sequences and sets of 6 CDR sequences as present in thepolypeptides of the invention and outlined above are summarized in thefollowing Tables I and II, respectively:

TABLE I and TABLE II: Preferred CDR combinations in polypeptides of theinvention comprising two different A-beta binding immunoglobulin singlevariable domains

TABLE I  CDR sequences SEQ ID NO: amino acid sequence 11 TDTMG 12AVTWNSGRTNYADSVKG 13 HRLVVGGTSVGDWRY 14 TDTMG 15 AVTWNSGRINYADSVKG 16HRFVVGGNRVEDWRY 17 NYNMG 18 AVSRSGVSTYYADSVKG 19 AYRGTAINVRRSYSS

TABLE II Preferred sets of CDR sequences/CDR combinations (sequencesdefined by their SEQ ID NO: as given in above TABLE I): set 1 set 2 set3 set 4 CDR(1)1 11 14 17 17 CDR(1)2 12 15 18 18 CDR(1)3 13 16 19 19CDR(2)1 17 17 11 14 CDR(2)2 18 18 12 15 CDR(2)3 19 19 13 16

Human immunoglobulin framework region sequences (FR) that can also beused as framework region sequences for the immunoglobulin singlevariable domains as described above are known in the art. Also known inthe art are methods for humanizing framework regions of immunoglobulinsingle variable domains derived from species other than humans.

In a preferred embodiment, the polypeptides of the invention comprisethe following framework region amino acid sequences 1 to 4 (FR1 to FR4;SEQ ID NOs as indicated in Table III below):

FR1 is or comprises an amino acid sequence selected from the groupconsisting of amino acid sequences according to SEQ ID NO:20 and SEQ IDNO:21;

FR2 is or comprises an amino acid sequence according to SEQ ID NO:22;

FR3 is or comprises an amino acid sequence selected from the groupconsisting of amino acid sequences according to SEQ ID NO:23 and SEQ IDNO:24; and

FR4 is or comprises an amino acid sequence according to SEQ ID NO:25.

TABLE II1  FR amino acid sequences SEQ ID NO: amino acid sequence 20VQLLESGGGLVQPGGSLRLSCVHSGPTFR 21 VQLLESGGGLVQPGGSLRLSCAASGRTFN 22WFRQAPGKGREFVA 23 RFTISRDNSKNTAYLQMNSLRPEDTAVYYCAA 24RFTISRDNSKNTVYLQMNSLRPEDTAVYYCAA 25 WGQGTLVTVSS

Specific examples of immunoglobulin single variable domains having theFR and CDR sequences as shown above are:

Immunoglobulin single variable domain 1 (first amino acid, i.e.glutamate, may optionally be missing):evqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefyaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtivtvss (ABII035; SEQ ID NO:44)

Immunoglobulin single variable domain 2 (first amino acid, i.e.glutamate, may optionally be missing):evqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefyaaysrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtivtvss (ABII059; SEQ ID NO:45)

Specific examples of polypeptides of the invention which include, in onesingle polypeptide chain, two immunoglobulin single variable domains asshown above and, optionally, a linker which connects the twoimmunoglobulin single variable domains, are given further below.

According to a preferred embodiment, the polypeptides of the inventioninclude, especially when used as a therapeutic agent, a moiety whichextends the half-life of the polypeptide of the invention in serum orother body fluids of a patient. The term “half-life” means the timetaken for the serum concentration of the (modified) polypeptide toreduce by 50%, in vivo, for example due to degradation of thepolypeptide and/or clearance and/or sequestration by natural mechanisms.

More specifically, such half-life extending moiety can be covalentlylinked or fused to said polypeptide and may be, without limitation, anFc portion, an albumin moiety, a fragment of an albumin moiety, analbumin binding moiety, such as an anti-albumin immunoglobulin singlevariable domain, a transferrin binding moiety, such as ananti-transferrin immunoglobulin single variable domain, apolyoxyalkylene molecule, such as a polyethylene glycol molecule, analbumin binding peptide, or hydroxyethyl starch (HES) derivatives.

According to one embodiment, the polypeptide of the invention may belinked to one or more antibody parts, fragments or domains that conferone or more effector functions to the polypeptide of the inventionand/or may confer the ability to bind to one or more Fc receptors. Forexample, for this purpose, and without being limited thereto, theantibody parts may be or may comprise CH2 and/or CH3 domains of anantibody, such as from a heavy chain antibody (as described hereabove)and more preferably from a conventional human 4-chain antibody;specifically, the polypeptide of the invention may be linked to an Fcregion, for example from human IgG, from human IgE or from another humanIg. For example, WO 94/04678 describes heavy chain antibodies comprisinga Camelid VHH domain or a humanized derivative thereof, in which theCamelidae CH2 and/or CH3 domain have been replaced by human CH2 and/orCH3 domains, so as to provide an immunoglobulin that consists of 2 heavychains each comprising a—optionally humanized—VHH domain and human CH2and CH3 domains (but no CH1 domain), which immunoglobulin has theeffector function provided by the CH2 and CH3 domains, can functionwithout the presence of any light chains, and has an increased half-lifeas compared to the corresponding VHH domains without such modification.

Specific examples of polypeptides of the invention including an Fcportion (with or without effector functions) are the polypeptidesindicated hereinbelow:

(SEQ ID NO: 26)evqllesggglyqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlvtvssggggsgggsevqllesggglyqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlgssglyslssvvtvpssslgtqtyicnynhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk(SEQ ID NO: 27)evqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssgggsggggsggggsggggsggggsgggevqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlgssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcyvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnyfscsvmhealhnhytqkslslspgk (SEQ ID NO: 28)evqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssggggsggggsggggcggggsggggsggggsggggsevqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlytvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnynhkpsntkvdkrvepkscdkthtcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID NO: 29)evqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlvtvssggggsgggsevqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlgssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk(SEQ ID NO: 30)evqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssgggsggggsggggsggggsggggsgggevqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreegynstyrvvsvltylhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesnggpennykttppyldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID NO: 31)evqllesggglvqpggslrlscaasgrtfnnynmgwfrqapgkgrefvaavsrsgvstyyadsvkgrftisrdnskntvylqmnslrpedtavyycaaayrgtainvrrsysswgqgtlvtvssggggsggggsggggcggggsggggsggggsggggsevqllesggglvqpggslrlscvhsgptfrtdtmgwfrqapgkgrefvaavtwnsgrinyadsvkgrftisrdnskntaylqmnslrpedtavyycaahrfvvggnrvedwrywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgyhtfpavlgssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdkthtcppcpapeaaggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreegynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppyldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk

According to a further embodiment of the invention, the twoimmunoglobulin single variable domains may be fused to a serum albuminmolecule, such as described e.g. in WO01/79271 and WO03/59934.

An example of a biparatopic A-beta binding polypeptide of the inventioncomprising a human serum albumin moiety is given in Table IV.

TABLE IV  HSA-fusion protein SEQ ID Sequence information Description NO:EVQLLESGGGLVQPGGSLRLSCAASGRTFNN 059-27GS- 32YNMGWFRQAPGKGREFVAAVSRSGVSTYYAD 035-HSA SVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS GGGSGGGGSGGGGSGGGGSGGGGSGGGEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG WFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCA AHRFVVGGNRVEDWRYWGQGTLVTVSSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCP FEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPE RNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAK RYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQ RFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPL LEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVL LLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNAL LVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

In another preferred embodiment, the polypeptide of the inventioncomprises a moiety which binds to an antigen found in blood, such asserum albumin, serum immunoglobulins, thyroxine-binding protein,fibrinogen or transferrin, thereby conferring an increased half-life invivo to the resulting polypeptide of the invention. According to aspecifically preferred embodiment, such moiety is an albumin-bindingimmunoglobulin and, especially preferred, an albumin-bindingimmunoglobulin single variable domain such as an albumin-binding VHHdomain.

If intended for use in humans, such albumin-binding immunoglobulinsingle variable domain will preferably bind to human serum albumin andwill preferably be a humanized albumin-binding VHH domain.

Immunoglobulin single variable domains binding to human serum albuminare known in the art and are described in further detail in e.g.WO2006/122786. A specifically useful albumin binding VHH domain consistsof or contains the amino acid sequence:

(SEQ ID NO: 33)evqlvesggglvqpgnslrlscaasgftfssfgmswvrqapgkglewvssisgsgsdtlyadsvkgrftisrdnakttlylqmnslrpedtavyyctiggslsrssqgtlvtvss

Specific examples of polypeptides of the invention which comprise analbumin binding VHH domain are shown in Table V:

TABLE V  Biparatopic polypeptides of the invention, comprising twoanti-A-beta VHH domains and one anti-HSA VHH domain SEQ CloneSequence information Description ID NO: ABII316EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-9GS- 34WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS Alb8-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR 035RSYSSWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGG GGSGGGSEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHR FVVGGNRVEDWRYWGQGTLVTVSS ABII317EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-35GS- 35WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS 035-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR Alb8RSYSSWGQGTLVTVSSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS GGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG GSLSRSSQGTLVTVSS ABII318EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-9GS- 36WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS 035-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR Alb8RSYSSWGQGTLVTVSSGGGGSGGGSEVQLLESGGG LVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQ MNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSL RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSS ABII319EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-35GS- 37WFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTIS 059-9GS-RDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNR Alb8VEDWRYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVS SGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG GSLSRSSQGTLVTVSS ABII320EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGW 035-9GS- 38FRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRD Alb8-9GS-NSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVE 059DWRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNY NMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAI NVRRSYSSWGQGTLVTVSS ABII321VQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWF 035-9GS- 39RQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDN Alb8-9GS-SKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVED 059WRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLV (first EQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEW deleted)VSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINV RRSYSSWGQGTLVTVSS

In still another preferred embodiment, the polypeptide of the inventioncomprises a moiety which binds to serum albumin, wherein such moiety isan albumin binding peptide, as described e.g. in international patentpublications WO2008/068280 and WO2009/127691.

According to still another embodiment, a half-life extendingmodification of a polypeptide of the invention (such modification alsoreducing immunogenicity of the polypeptide) comprises attachment of asuitable pharmacologically acceptable polymer, such as straight orbranched chain poly(ethylene glycol) (PEG) or derivatives thereof (suchas methoxypoly(ethylene glycol) or mPEG). Generally, any suitable formof PEGylation can be used, such as the PEGylation used in the art forantibodies and antibody fragments (including but not limited to domainantibodies and scFv's); reference is made, for example, to: Chapman,Nat. Biotechnol., 54, 531-545 (2002); Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003); Harris and Chess, Nat. Rev. Drug.Discov. 2 (2003); WO 04/060965; and U.S. Pat. No. 6,875,841.

Various reagents for PEGylation of polypeptides are also commerciallyavailable, for example from Nektar Therapeutics, USA, or NOFCorporation, Japan, such as the Sunbright® EA Series, SH Series, MASeries, CA Series, and ME Series, such as Sunbright® ME-100MA,Sunbright® ME-200MA, and Sunbright® ME-400MA.

Preferably, site-directed PEGylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering 16,761-770 (2003)). For example, for this purpose, PEG may be attached to acysteine residue that naturally occurs in a polypeptide of theinvention, a polypeptide of the invention may be modified so as tosuitably introduce one or more cysteine residues for attachment of PEG,or an amino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus and/or of alinker region that bridges two or more functional domains of apolypeptide of the invention, all using techniques of proteinengineering known per se to the skilled person.

Preferably, for the polypeptides of the invention, a PEG is used with amolecular weight of more than 5 kDa, such as more than 10 kDa and lessthan 200 kDa, such as less than 100 kDa; for example in the range of 20kDa to 80 kDa.

With regard to PEGylation, it should be noted that generally, theinvention also encompasses any polypeptide of the invention that hasbeen PEGylated at one or more amino acid positions, preferably in such away that said PEGylation either (1) increases the half-life in vivo; (2)reduces immunogenicity; (3) provides one or more further beneficialproperties known per se for PEGylation; (4) does not essentially affectthe affinity of the polypeptide for A-beta (e.g. does not reduce saidaffinity by more than 50%, and more preferably not by more than 10%, asdetermined by a suitable assay, such as those described in the Examplesbelow); and/or (4) does not affect any of the other desired propertiesof the polypeptides of the invention. Suitable PEG-groups and methodsfor attaching them, either specifically or non-specifically, will beclear to the skilled person.

According to a specifically preferred embodiment of the invention, aPEGylated polypeptide of the invention includes one PEG moiety of linearPEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEGmoiety is attached to the polypeptide in a linker region and,specifically, at a Cys residue at position 5 of a GS9-linker peptide asshown in SEQ ID NO:6, at position 14 of a GS27-linker peptide as shownin SEQ ID NO:8, or at position 15 of a GS35-linker peptide as shown inSEQ ID NO:9, or at position 5 of a 35GS-linker peptide as shown in SEQID NO:10.

Preferred examples of polypeptides of the invention, PEGylatedpreferably with one of the PEG reagents as mentioned above, such as“Sunbright® ME-400MA” as shown in the following chemical formula:

which has an average molecular weight of 40 kDa, are given in Table VIbelow:

TABLE VI  PEGylated polypeptides of the invention; C* indicates theCys residue bearing the PEG moiety SEQ ID Clone Sequence informationDescription NO: ABII322 VQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFR ABII059-40 QAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSK 27GSNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSW linkerGQGTLVTVSSGGGSGGGGSGGGGC*GGGGSGGGGS with C atGGGEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM positionGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISR 14-DNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVE ABII035 DWRYWGQGTLVTVSS ABII323VQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFR ABII059- 41QAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSK 35GSNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSW linkerGQGTLVTVSSGGGGSGGGGSGGGGC*GGGGSGGGG with C atSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCVHS positionGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYAD 15-SVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHR ABII035 FVVGGNRVEDWRYWGQGTLVTVSSABII305 EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYNMGWF ABII050- 42RQAPGKEREFVAAVSRSGVSTYYADSVKGRFTISRDNA 35GSKNAVYLQMNSLKPEDTAIYYCGAAYRGTAINVRRSYDS linkerWGQGTQVTVSSGGGGC*GGGGSGGGGSGGGGSGGG with C atGSGGGGSGGGGSEVQLVESGGGLVLAGGSLRLSCVH positionSGPTFRTDTMGWFRQAPGKEREFVAAVTWNSGRINYA 5-DSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTA ABII002 HRFVVGGNRVEDWRYWGQGTQVTVSSABII306 EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYNMGWF ABII050- 43RQAPGKEREFVAAVSRSGVSTYYADSVKGRFTISRDNA 9GSKNAVYLQMNSLKPEDTAIYYCGAAYRGTAINVRRSYDS linkerWGQGTQVTVSSGGGGC*GGGSEVQLVESGGGLVLAG with C atGSLRLSCVHSGPTFRTDTMGWFRQAPGKEREFVAAVT positionWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSGLKDE 5-DTAVYYCTAHRFVVGGNRVEDWRYWGQGTQVTVSS ABII002 ABII314EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWF ABII059- 142RQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNS 35GSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSS linkerWGQGTLVTVSSGGGGC*GGGGSGGGGSGGGGSGGG with C atGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCVHS positionGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYAD 5-SVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHR ABII035 FVVGGNRVEDWRYWGQGTLVTVSSABII315 EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWF ABII059- 143RQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNS 9GSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSS linkerWGQGTLVTVSSGGGGC*GGGSEVQLLESGGGLVQPGG with C atSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTW positionNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTA 5-VYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII035

According to a further embodiment, the polypeptide of the inventionadditionally comprises a moiety which allows the polypeptide to crossthe blood brain barrier. In particular, said moiety that allows theresulting polypeptides of the invention to cross the blood brain barriermay be one or more (such as two and preferably one) immunoglobulinsingle variable domains such as the brain targeting antibody fragments(VHHs) FC44 and FC5 described in WO02/057445. Examples thereof are shownin Table XIX below.

Thus, the polypeptides of the invention can also be described by thefollowing general formula:A-ISVD1-B-ISVD2-C, whereinISVD1 and ISVD2 denote an A-beta binding immunoglobulin single variabledomain as described hereinbefore, binding to different epitopes ofA-beta, such as e.g. the immunoglobulin single variable domainsaccording to SEQ ID NOs 44 and 45, respectively; andA, B and C independently of each other denote:

-   -   no additional moiety (i.e. A is the N-terminal end of ISVD1, B        is a peptide bond linking ISVD1 and ISVD2, and C is the        C-terminal end of ISVD2)    -   one or more domains selected from the group of CH1, CH2, and CH3        domains of human IgG, IgM, IgD, IgE, and the like, and        preferably C denotes CH2-CH3;    -   albumin or a fragment thereof, and preferably human serum        albumin or a fragment thereof;    -   an albumin binding moiety, and preferably an albumin binding        immunoglobulin single variable domain or an albumin binding        peptide;    -   a linker peptide, which is optionally PEGylated; and preferably        B denotes a linker peptide having from 3 to 45 amino acids,        including at least one cysteine residue which is PEGylated,        preferably with a PEG40 or PEG60 moiety;    -   one or more A-beta binding moieties, preferably an additional        anti-A-beta immunoglobulin variable domain, identical or not        identical to ISVD1 or ISVD2    -   a polypeptide, preferably an immunoglobulin single variable        domain, conferring to the polypeptide blood-brain-barrier        crossing properties, such as FC44 and FC5 described in        WO02/057445;        or wherein        ISVD1, ISVD2, B and C have the meaning as above and    -   A denotes an N-terminal, optionally formylated, Met residue, for        example as result of expression in a heterologous host organism;        and/or a signal or leader sequence that directs secretion of the        polypeptide of the invention from a host cell upon synthesis;        and/or a pro-sequence which is optionally removed after        expression of the polypeptide in a suitable host cell;        or wherein        ISVD1, ISVD2, A and B have the meaning as above and    -   C denotes a “tag”, such as an amino acid sequence or residue        that allows or facilitates the purification of the polypeptide        of the invention, for example using affinity techniques directed        against said sequence or residue; such tag may optionally be        removable after such purification step, e.g. by chemical or        enzymatical cleavage, to provide the mature sequence of the        polypeptide; for this purpose, the tag may optionally be linked        to the polypeptide sequence via a cleavable linker sequence or        contain a cleavable motif. Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutathione residues and a myc-tag such as AAAEQKLISEEDLNGAA        (SEQ ID NO:46).        Therapeutic Use

According to an important further aspect, the polypeptide of theinvention is used for therapeutic purposes, such as

-   -   for the prevention, treatment and/or alleviation of a disorder,        disease or condition as set out below, especially in a human        being, such as Alzheimer's Disease (AD), dry AMD, or glaucoma;    -   in a method of treatment of a patient in need of such therapy,        such method comprising administering, to the subject in need        thereof, a pharmaceutically active amount of at least one        polypeptide of the invention or a pharmaceutical composition (as        set out in detail below) comprising such polypeptide of the        invention, wherein such subject in need of such therapy may be a        human being suffering from any of the disorders, diseases or        conditions as set out below, such as AD, dry AMD, or glaucoma;    -   for the preparation of a medicament for the prevention,        treatment or alleviation of disorders, diseases or conditions as        set out below, such as AD, dry AMD, or glaucoma in a human        being;    -   as an active ingredient in a pharmaceutical composition or        medicament used for the before-mentioned purposes.

The disorder, disease or condition (in the following: disease) asmentioned above is a disease that can be prevented and/or treated and/oralleviated by administering a polypeptide of the invention to thepatient or (human) being, and, more specifically, a disease mediated byA-beta dysfunction, such as a dysfunction of A-beta production,deposition or lack of clearance, and/or a disease mediated by amyloidplaque formation, formation of A-beta oligomers, and the like. Even morespecifically, the disease is a disease that can be prevented and/ortreated and/or alleviated by modulating, reducing and/or reversing the(undesired) formation or build-up of A-beta and/or of amyloid plaquesand/or of A-beta oligomers in a patient, such diseases comprising e.g.neurodegenerative diseases, the most prominent A-beta relatedneurodegenerative disease being AD.

Thus, the polypeptides of the invention can be used in a method for theprevention, treatment or alleviation of diseases such as:

-   -   Alzheimer's disease (AD; all types and stages of the disease        including preclinical and prodromal stages); also known as        “dementia of the Alzheimer type”;    -   the dry form of age-related macular degeneration (dry AMD;        central geographic atrophy)    -   glaucoma    -   cerebral amyloid angiopathy (CAA);

trisomy 21 (Down's Syndrome), including adult Down syndrome;

-   -   hereditary cerebral hemorrhage with amyloidosis of the        Dutch-type (HCHWA-D);    -   dementia with Lewy Bodies;    -   frontotemporal lobar degeneration;    -   glaucoma;    -   amyotrophic lateral sclerosis;    -   sporadic inclusion body myositis; and    -   anxiety disorder in an elderly human subject (e.g. of at least        55 years old), selected from the group consisting of        obsessive-compulsive disorder, panic disorder, panic attack,        agoraphobia, post-traumatic stress disorder, social phobia,        disruptive behaviour disorder and chronic fatigue syndrome        (wherein said elderly human subject may or may not be diagnosed        with a condition related to A-beta, selected from clinical or        preclinical AD, chronic amyloid angiopathy and Down's syndrome);        wherein such method comprises administering, to a subject in        need thereof, a pharmaceutically active amount of a polypeptide        of the invention and/or of a pharmaceutical composition        comprising the same.

In the context of the present invention, the term “prevention, treatmentand/or alleviation” not only comprises preventing and/or treating and/oralleviating the disease, but also generally comprises preventing theonset of the disease, slowing or reversing the progress of disease,preventing or slowing the onset of one or more symptoms associated withthe disease, reducing and/or alleviating one or more symptoms associatedwith the disease, reducing the severity and/or the duration of thedisease and/or of any symptoms associated therewith and/or preventing afurther increase in the severity of the disease and/or of any symptomsassociated therewith, preventing, reducing or reversing anyphysiological damage caused by the disease, and generally anypharmacological action that is beneficial to the patient being treated.

Specifically, in the case of AD, the polypeptides, compositions, andmethods of the invention can be used for helping to prevent or delay theonset of AD in patients with identified risk factors for AD and/orproven A-beta deposits, for treating patients with mild cognitiveimpairment (MCI), who are at risk to convert to AD, and for preventingor delaying the onset of AD in those patients who would otherwise beexpected to progress from preclinical or prodromal disease stages todementia. Genetic risk factors for AD include specific mutations in theAPP gene (in particular at positions 670 and 671 as well as position717), in the presenilin genes PS1 and PS2, and in ApoE4. Other riskfactors include a family history of AD, hypercholesterolemia,atherosclerosis, diabetes, and/or high age. Disposition for AD can bediagnosed early by analysing A-beta(1-42) and tau concentrations in CSF.Neuroimaging techniques (e.g. PET and MRI) may identify patients at riskof converting to AD. In general, the use of several biomarkers may allowto diagnose AD in a very early stage with a high sensitivity andspecificity.

In the case of a predisposition for Down's syndrome, hereditary cerebralhemorrhage with amyloidosis of the Dutch type, and cerebral beta-amyloidangiopathy, the polypeptides, compositions, and methods of the inventionmay also be useful for preventing their potential consequences such assingle and recurrent lobar hemorrhages.

The subject to be treated will be a mammal, and more in particular ahuman being. As will be clear to the skilled person, the subject to betreated will in particular be a person suffering from, or at risk from,the diseases, disorders or conditions mentioned herein.

It will also be clear to the skilled person that the above methods oftreatment of a disease include the preparation of a medicament for thetreatment of said disease. Furthermore, it is clear that thepolypeptides of the invention can be used as an active ingredient in amedicament or pharmaceutical composition intended for the treatment ofthe above diseases. Thus, the invention also relates to the use of apolypeptide of the invention in the preparation of a pharmaceuticalcomposition for the prevention, treatment and/or alleviation of any ofthe diseases, disorders or conditions mentioned hereinabove. Theinvention further relates to a polypeptide of the invention fortherapeutic or prophylactic use and, specifically, for the prevention,treatment and/or alleviation of any of the diseases, disorders orconditions mentioned hereinabove. The invention further relates to apharmaceutical composition for the prevention, treatment and/oralleviation of the diseases, disorders or conditions mentionedhereinabove, wherein such composition comprises at least one polypeptideof the invention.

Without wishing to be bound by a specific theory, the above therapeuticor prophylactic effect may be effected by the following mechanism: Thepolypeptides of the invention bind to A-beta, thereby inhibiting itsinteraction with one or more other A-beta molecules or the interactionof A-beta with a receptor or the interaction of A-beta with a solublebiomolecule or the interaction of A-beta with an insoluble biomolecule.The target A-beta may be a part of a plaque or suspension or solution,or one or more of these, wherein the other A-beta molecules may also bea part of a plaque, in suspension or solution or one or more of these.Clearance of different A-beta forms, such as the monomeric form, oligo-and multimeric forms, aggregated soluble and insoluble forms, fibrillarforms, proto-fibrillar forms and amyloid plaques from the brain, bloodvessels or other parts in the body may be due to the binding of thepolypeptide of the invention to A-beta. Reduction of A-beta levels in abody fluid, and preferably the reduction of the level of soluble A-betain blood by inhibition of the interaction between an A-beta molecule andanother molecule, such as e.g. another A-beta molecule, will alleviatethe symptoms of degenerative neural diseases, slow down or stop thedisease progression and/or restore brain damage, memory and cognition.

According to a further, more general aspect, the invention relates to amethod for immunotherapy, and in particular for passive immunotherapy,which method comprises administering, to a subject suffering from or atrisk of the diseases as mentioned herein, a pharmaceutically activeamount of a polypeptide of the invention and/or of a pharmaceuticalcomposition comprising the same.

According to still another aspect, the invention relates to a method (i)for the prevention, treatment and/or alleviation of cognitive decline,and/or (ii) for restoring cognitive function and/or of improvingcognitive function, said method comprising administering, to a subjectin need thereof, a pharmaceutically active amount of a polypeptide ofthe invention and/or of a pharmaceutical composition comprising thesame.

The polypeptides of the invention and/or the compositions comprising thesame can be administered to a patient in need thereof in any suitablemanner, depending on the specific pharmaceutical formulation orcomposition to be used. Thus, the polypeptides of the invention and/orthe compositions comprising the same can for example be administeredintravenously, subcutaneously, intramuscularly, intraperitoneally,transdermally, orally, sublingually (e.g. in the form of a sublingualtablet, spray or drop placed under the tongue and adsorbed through themucus membranes into the capillary network under the tongue),(intra-)nasally (e.g. in the form of a nasal spray and/or as anaerosol), topically, by means of a suppository, by inhalation,intravitreally (esp. for the treatment of dry AMD or glaucoma), or anyother suitable manner in an effective amount or dose.

The polypeptides of the invention and/or the compositions comprising thesame are administered according to a regimen of treatment that issuitable for preventing, treating and/or alleviating the disease,disorder or condition to be prevented, treated or alleviated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease, disorder or conditionto be prevented, treated or alleviated, the severity of the disease, theseverity of the symptoms thereof, the specific polypeptide of theinvention to be used, the specific route of administration andpharmaceutical formulation or composition to be used, the age, gender,weight, diet, general condition of the patient, and similar factors wellknown to the clinician. Generally, the treatment regimen will comprisethe administration of one or more polypeptides of the invention, or ofone or more compositions comprising the same, in therapeutically and/orprohylactically effective amounts or doses.

Generally, for the prevention, treatment and/or alleviation of thediseases, disorders and conditions mentioned herein and depending on thespecific disease, disorder or condition to be treated, the potency ofthe specific polypeptide of the invention to be used, the specific routeof administration and the specific pharmaceutical formulation orcomposition used, the polypeptides of the invention will generally beadministered in an amount between 0.005 and 20.0 mg per kilogram of bodyweight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and morepreferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. byinfusion) or as single doses (such as e.g. daily, weekly, or monthlydoses; cf. below), but can significantly vary, especially, depending onthe before-mentioned parameters.

For prophylactic applications, compositions containing the polypeptidesof the invention may also be administered in similar or slightly lowerdosages. The dosage can also be adjusted by the individual physician inthe event of any complication.

Depending on the specific polypeptide of the invention and its specificpharmacokinetic and other properties, it may be administered daily,every second, third, fourth, fifth or sixth day, weekly, monthly, andthe like. An administration regimen could include long-term, weeklytreatment. By “long-term” is meant at least two weeks and preferablymonths, or years of duration.

The efficacy of the polypeptides of the invention, and of compositionscomprising the same, can be tested using any suitable in vitro assay,cell-based assay, in vivo assay and/or animal model known per se, or anycombination thereof, depending on the specific disease involved.Suitable assays and animal models will be clear to the skilled person,and for example include the assays and animal models used in theExamples below. Thus, suitable assays are, but are not limited to, ELISAbinding assays measuring the binding of the anti-A-beta polypeptides tocoated monomeric or aggregated A-beta peptides or to capturedbiotinylated A-beta, SPR (Surface Plasmon Resonance) assays measuringthe binding to coated monomeric or aggregated A-beta peptides or tocaptured biotinylated A-beta (Malmqvist M.: Surface plasmon resonancefor detection and measurement of antibody-antigen affinity and kinetics;Curr. Opin. Immunol. 5(2):282 (1993)), competition TR-FRET (TimeResolved Fluorescence Resonance Energy Transfer) assays measuring thecompetition with either an A-beta(1-40) peptide/N-terminal regionspecific binder interaction or with an A-beta(1-40)/central regionspecific binder interaction, in vitro A-beta aggregation assaysmeasuring the prevention or disaggregation of the aggregation, TAPIR(Tissue Amyloid Plaque Immunoreactivity) assays measuring the binding ofmolecules to amyloid plaques using immunohistochemical analysis onbrains from Alzheimer's disease patients or APP transgenic animals, aswell as in vivo mechanistic models.

Preferably, the polypeptides of the invention are having bettercharacteristics than conventional antibodies known in the art (such asm266 and 3D6 anti-A-beta IgG antibodies) or the Nanobodies® described inWO2006/40153 in at least one of these assays or models, and preferablyin one or more of the in vivo models.

For pharmaceutical use, the polypeptides of the invention may beformulated as a pharmaceutical preparation comprising (i) at least onepolypeptide of the invention and (ii) at least one pharmaceuticallyacceptable carrier, diluent, excipient, adjuvant, and/or stabilizer, and(iii) optionally one or more further pharmaceutically activepolypeptides and/or compounds. By “pharmaceutically acceptable” is meantthat the respective material does not show any biological or otherwiseundesirable effects when administered to an individual and does notinteract in a deleterious manner with any of the other components of thepharmaceutical composition (such as e.g. the pharmaceutically activeingredient) in which it is contained. Specific examples can be found instandard handbooks, such as e.g. Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990). For example, thepolypeptides of the invention may be formulated and administered in anymanner known per se for conventional antibodies and antibody fragmentsand other pharmaceutically active proteins. Thus, according to a furtherembodiment, the invention relates to a pharmaceutical composition orpreparation that contains at least one polypeptide of the invention andat least one pharmaceutically acceptable carrier, diluent, excipient,adjuvant and/or stabilizer, and optionally one or more furtherpharmaceutically active substances.

By means of non-limiting examples, such a formulation may be in a formsuitable for oral administration, for parenteral administration (such asby intravenous, intramuscular, subcutaneous, intrathecal,intracavernosal or intraperitoneal injection or intravenous infusion),for topical administration, for sublingual administration, foradministration by inhalation, by a skin patch, by an implant, by asuppository, for transdermal, nasal, intravitreal, rectal or vaginaladministration, and the like. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person.

Pharmaceutical preparations for parenteral administration, such asintravenous, intramuscular, subcutaneous injection or intravenousinfusion may for example be sterile solutions, suspensions, dispersions,emulsions, or powders which comprise the active ingredient and which aresuitable, optionally after a further dissolution or dilution step, forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, sterile water andpharmaceutically acceptable aqueous buffers and solutions such asphysiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution; water oils; glycerol; ethanol; glycolssuch as propylene glycol, as well as mineral oils, animal oils andvegetable oils, for example peanut oil, soybean oil, as well as suitablemixtures thereof.

Solutions of the active compound or its salts may also contain apreservative to prevent the growth of microorganisms, such asantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and thelike. In many cases, it will be preferable to include isotonic agents,for example, sugars, buffers or sodium chloride. The proper fluidity canbe maintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. Other agents delaying absorption, forexample, aluminum monostearate and gelatin, may also be added.

In all cases, the ultimate dosage form must be sterile, fluid and stableunder the conditions of manufacture and storage. Sterile injectablesolutions are prepared by incorporating the active compound in therequired amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

Usually, aqueous solutions or suspensions will be preferred. Generally,suitable formulations for therapeutic proteins such as the polypeptidesof the invention are buffered protein solutions, such as solutionsincluding the protein in a suitable concentration (such as from 0.001 to400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to200 mg/ml, more preferably 1.0-100 mg/ml, such as 1.0 mg/ml (i.v.administration) or 100 mg/ml (s.c. administration) and an aqueous buffersuch as:

-   -   phosphate buffered saline, pH 7.4,    -   other phosphate buffers, pH 6.2 to 8.2,    -   histidine buffers, pH 5.5 to 7.0,    -   succinate buffers, pH 3.2 to 6.6, and    -   citrate buffers, pH 2.1 to 6.2,        and, optionally, salts (e.g. NaCl) and/or sugars or polyalcohols        (such as trehalose, mannitol, or glycerol) for providing        isotonicity of the solution.

Preferred buffered protein solutions are solutions including about 0.05mg/ml of the polypeptide of the invention dissolved in 25 mM phosphatebuffer, pH 6.5, adjusted to isotonicity by adding 220 mM trehalose. Inaddition, other agents such as a detergent, e.g. 0.02% Tween-20 orTween-80, may be included in such solutions. Formulations forsubcutaneous application may include significantly higher concentrationsof the polypeptide of the invention, such as up to 100 mg/ml or evenabove 100 mg/ml. However, it will be clear to the person skilled in theart that the ingredients and the amounts thereof as given above do onlyrepresent one, preferred option. Alternatives and variations thereofwill be immediately apparent to the skilled person, or can easily beconceived starting from the above disclosure.

The polypeptides of the invention may also be administered usingsuitable depot, slow-release or sustained-release formulations, e.g.suitable for injection, using controlled-release devices forimplantation under the skin, and/or using a dosing pump or other devicesknown per se for the administration of pharmaceutically activesubstances or principles. In addition, the polypeptides of the inventionmay be formulated in the form of a gel, cream, spray, drop, patch orfilm which, if placed on the skin, passes through the skin.

Also, compared to conventional antibodies or antibody fragments, onemajor advantage of the use of the polypeptides of the invention is thatthey can also be easily administered via routes other than parenteraladministration and can be easily formulated for such administration. Forexample, as described in the international application WO2004/041867,such polypeptides may be formulated for oral, intranasal, intrapulmonaryand transdermal administration.

According to another embodiment of the invention there is provided apharmaceutical combination comprising at least one anti-A-betapolypeptide of the invention as disclosed herein and at least one othertherapeutic agent selected from the group consisting of: cholinesteraseinhibitors such as donepezil, rivastigmine, galantamine, and tacrine;NMDA antagonists such as memantine; A-beta lowering agents such asagents capable of inhibiting one or more enzymes involved in formationof A-beta, such as beta-secretase inhibitors, gamma-secretase inhibitorsand gamma-secretase modulators; agents that prevent or reduce A-betaplaque building; A-beta aggregation inhibitors; RAGE antagonists; andother agents for preventing, treating or alleviating neurodegenerativediseases and/or decline in cognitive function. Specific examples of suchother therapeutic agents are: ELND-005, Caprospinol, NRM-8499, PBT-2,Posiphen, EHT-0202, CTS-21166, Semagacest, BMS-708163, BMS-299897,BMS-433796, ELND-006, ELN-475516, ELN-318463, ELN-475513, Begacestat,E-2012, CHF-5074, Dimebolin (Latrepiridin), and PF-4494700. Suchpharmaceutical combination may optionally additionally comprise adiluent, excipient, adjuvant and/or stabilizer.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition. Also, when two ormore active substances or principles are to be used as part of acombined treatment regimen, each of the substances or principles may beadministered in the same amount and according to the same regimen asused when the compound or principle is used on its own, and suchcombined use may or may not lead to a synergistic effect. However, whenthe combined use of the two or more active substances or principlesleads to a synergistic effect, it may also be possible to reduce theamount of one, more or all of the substances or principles to beadministered, while still achieving the desired therapeutic action. Thismay for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

Yet a further embodiment of the invention is a method for treating thediseases and disorders as set out above, comprising administering to anindividual, simultaneously, separately or sequentially, an effectiveamount of at least one anti-A-beta polypeptide of the invention and atleast one agent selected from the group consisting of: cholinesteraseinhibitors such as donepezil, rivastigmine, galantamine, and tacrine;NMDA antagonists such as memantine; A-beta lowering agents such asagents capable of inhibiting one or more enzymes involved in formationof A-beta, such as beta-secretase inhibitors, gamma-secretase inhibitorsand gamma-secretase modulators; agents that prevent or reduce A-betaplaque building; A-beta aggregation inhibitors; RAGE antagonists; andother agents for preventing, treating or alleviating neurodegenerativediseases and/or decline in cognitive function, including the specificexamples as set out above.

According to a further aspect of the invention, the A-beta bindingpolypeptide of the invention is prepared to be administered incombination with other drugs used for the treatment of the diseases anddisorders set out above, such other drugs being selected from the groupconsisting of: cholinesterase inhibitors such as donepezil,rivastigmine, galantamine, and tacrine; NMDA antagonists such asmemantine; A-beta lowering agents such as agents capable of inhibitingone or more enzymes involved in formation of A-beta, such asbeta-secretase inhibitors, gamma-secretase inhibitors andgamma-secretase modulators; agents that prevent or reduce A-beta plaquebuilding; A-beta aggregation inhibitors; RAGE antagonists; and otheragents for preventing, treating or alleviating neurodegenerativediseases and/or decline in cognitive function, including the specificexamples as set out above.

According to still another aspect of the invention, drugs used for thetreatment of the diseases and disorders set out above, such drugs beingselected from the group consisting of: cholinesterase inhibitors such asdonepezil, rivastigmine, galantamine, and tacrine; NMDA antagonists suchas memantine; A-beta lowering agents such as agents capable ofinhibiting one or more enzymes involved in formation of A-beta, such asbeta-secretase inhibitors, gamma-secretase inhibitors andgamma-secretase modulators; agents that prevent or reduce A-beta plaquebuilding; A-beta aggregation inhibitors; RAGE antagonists; and otheragents for preventing, treating or alleviating neurodegenerativediseases and/or decline in cognitive function (including the specificexamples as set out above) are prepared to be administered incombination with the A-beta binding polypeptide of the invention.

According to a further aspect of the invention, the A-beta bindingpolypeptide of the invention is used in combination with a device usefulfor the administration of the polypeptide, such as a syringe, injectorpen, or other device.

According to still another embodiment of the invention, there isprovided a method of diagnosing a disease, disorder or conditionmediated by A-beta dysfunction and/or amyloid plaque formationcomprising the steps of:

a) obtaining a sample from a subject, and

b) contacting, in vitro, the sample with a polypeptide of the inventionas defined above, and

c) detecting the binding of said polypeptide to said sample, and

d) comparing the binding detected in step (c) with a standard, wherein adifference in binding relative to said sample is diagnostic of adisease, disorder or condition characterised by A-beta dysfunctionand/or amyloid plaque formation.

According to another embodiment of the invention, there is provided amethod of diagnosing a disease, disorder or condition mediated by A-betadysfunction and/or amyloid plaque formation comprising the steps of:

a) obtaining a sample from a subject, and

b) contacting the sample with a polypeptide of the invention as definedabove;

c) determining the amount of A-beta in the sample; and

d) comparing the amount determined in step (c) with a standard, whereina difference in amount relative to said sample is diagnostic of adisease, disorder or condition characterised by A-beta dysfunctionand/or amyloid plaque formation.

The sample may e.g. be a body fluid of the subject, such as blood orcerebrospinal fluid (CSF). The step of detecting binding of apolypeptide of the invention to A-beta or the step of determining theamount of A-beta in the sample will generally involve measuring theformation of a complex between the polypeptide and A-beta. According todifferent embodiments of this method, complex formation will occur insolution or after immobilization of one component on a substrate andwill be followed by detection of such complexes. For this purpose, itmay be useful to further modify the polypeptide of the invention, suchas by introduction of a functional group that is one part of a specificbinding pair, such as the biotin-(strept)avidin binding pair. Such afunctional group may be used to link the polypeptide of the invention toanother protein, polypeptide or chemical compound that is bound to theother half of the binding pair, i.e. through formation of the bindingpair. For example, a polypeptide of the invention may be conjugated tobiotin, and linked to another protein, polypeptide, compound or carrierconjugated to avidin or streptavidin. For example, such a conjugatedpolypeptide of the invention may be used as a reporter, for example in adiagnostic system where a detectable signal-producing agent isconjugated to avidin or streptavidin.

The above diagnostic methods can also be used for monitoring theeffectiveness of a therapeutic treatment of a subject.

According to another embodiment of the invention, there is provided akit for diagnosing a disease, disorder or condition mediated by A-betadysfunction, and especially amyloid plaque formation and/or AD, for usein a method as defined above, such kit comprising at least onepolypeptide of the invention and, optionally, one or more media,detection means and/or in vitro or in vivo imaging agents, and, furtheroptionally, instructions of use. Suitable in vivo imaging agents include99 mTc, 111 Indium, 123 Iodine, and, for magnetic resonance imaging,paramagnetic compounds. The combination of SPECT, PET or MRI withlabeled anti-A-beta polypeptides of the invention will allow ‘A-betabrain scans’ and individual risk assessment for each patient.

According to still another embodiment of the invention, certain A-betabinding polypeptides of the invention can be used as a research tool forthe specific detection of human as well as mouse A-beta, or A-beta fromother animal species, and for tests and assays relying on suchdetection. This may be particularly useful for tests and assays makinguse of animal models.

The invention further provides a kit comprising at least one A-betabinding polypeptide of the invention and, additionally, one or moreother components selected from the group consisting of other drugs usedfor the treatment of the diseases and disorders as described above, anddevices as described above.

The invention further provides methods of manufacturing an A-betabinding polypeptide of the invention, such methods generally comprisingthe steps of:

-   -   culturing host cells comprising a nucleic acid capable of        encoding a polypeptide of the invention (hereinafter: “nucleic        acid of the invention”) under conditions that allow expression        of the polypeptide of the invention; and,    -   recovering or isolating the polypeptide expressed by the host        cells from the culture; and    -   optionally further purifying and/or modifying and/or formulating        the polypeptide of the invention.

A nucleic acid of the invention can be genomic DNA, cDNA or syntheticDNA (such as DNA with a codon usage that has been specifically adaptedfor expression in the intended host cell or host organism). According toone embodiment of the invention, the nucleic acid of the invention is inessentially isolated form, as defined hereabove.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form. The vector mayespecially be an expression vector, i.e. a vector that can provide forexpression of the polypeptide in vitro and/or in vivo (e.g. in asuitable host cell, host organism and/or expression system). Suchexpression vector generally comprises at least one nucleic acid of theinvention that is operably linked to one or more suitable regulatoryelement(s), such as promoter(s), enhancer(s), terminator(s), and thelike. Specific examples of such regulatory elements and other elements,such as integration factor(s), selection marker(s), signal or leadersequence(s), reporter gene(s), and the like, useful or necessary forexpressing polypeptides of the invention, are disclosed e.g. on pp. 131to 133 of WO2006/040153.

The nucleic acids of the invention can be prepared or obtained in amanner known per se (e.g. by automated DNA synthesis and/or recombinantDNA technology), based on the information on the amino acid sequencesfor the polypeptides of the invention given herein, and/or can beisolated from a suitable natural source.

According to another embodiment, the invention relates to a host or hostcell that expresses or is capable of expressing a polypeptide of theinvention; and/or that contains a nucleic acid encoding a polypeptide ofthe invention. According to a particularly preferred embodiment, saidhost cells are bacterial cells, yeast cells, fungal cells or mammaliancells.

Suitable bacterial cells include cells from gram-negative bacterialstrains such as strains of Escherichia coli, Proteus, and Pseudomonas,and gram-positive bacterial strains such as strains of Bacillus,Streptomyces, Staphylococcus, and Lactococcus. Suitable fungal cellinclude cells from species of Trichoderma, Neurospora, and Aspergillus.Suitable yeast cells include cells from species of Saccharomyces (forexample Saccharomyces cerevisiae), Schizosaccharomyces (for exampleSchizosaccharomyces pombe), Pichia (for example Pichia pastoris andPichia methanolica), and Hansenula.

Suitable mammalian cells include for example CHO cells, BHK cells, HeLacells, COS cells, and the like. However, amphibian cells, insect cells,plant cells, and any other cells used in the art for the expression ofheterologous proteins can be used as well.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of immunoglobulin single variable domainpolypeptides and protein therapeutics containing them include strains ofE. coli, Pichia pastoris, and S. cerevisiae that are suitable for largescale expression, production and fermentation, and in particular forlarge scale (bio-)pharmaceutical expression, production andfermentation.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of a polypeptide of theinvention for which glycosylation is desired or required wouldnecessitate the use of mammalian expression hosts that have the abilityto glycosylate the expressed protein. In this respect, it will be clearto the skilled person that the glycosylation pattern obtained (i.e. thekind, number and position of residues attached) will depend on the cellor cell line that is used for the expression.

Polypeptides of the invention produced in a cell as set out above can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or they can be produced extracellularly(secreted into the medium in which the host cells are cultured) and thenisolated from the culture medium and optionally further purified.

Further methods and reagents used for the recombinant production ofpolypeptides, such as suitable expression vectors, transformation ortransfection methods, selection markers, methods of induction of proteinexpression, culture conditions, and the like, are known in the art.Similarly, protein isolation and purification techniques useful in amethod of manufacture of a polypeptide of the invention are well knownto the skilled person.

Production of the polypeptides of the invention through fermentation inconvenient recombinant host organisms such as E. coli and yeast iscost-effective, as compared to conventional antibodies which alsorequire expensive mammalian cell culture facilities. Furthermore,achievable levels of expression are high and yields of the polypeptidesof the invention are in the range of 1 to 10 g/l (E. coli) and up to 10g/l (yeast) and more.

According to still another aspect of the invention, there are providedthe immunoglobulin single variable domains as listed in Table VII below,which are useful for building up or constructing A-beta bindingpolypeptides of the invention. Thus, if any of the immunoglobulin singlevariable domains as listed in Table VII (optionally after having beenhumanized) will be combined (preferably in the form of one continuouspolypeptide chain) with one or more other A-beta binding immunoglobulinsingle variable domain(s), wherein such other immunoglobulin singlevariable domain(s) bind(s) to a different epitope of A-beta, this willresult in biparatopic A-beta binding molecules according to theinvention, having useful binding characteristics, as set out in detaile.g. in Examples 9 to 11 below.

TABLE VII  A-beta binding immunoglobulin single variable domains IC50 inmelting SEQ TR-FRET temperature ID Clone (M) in ° C. amino acid sequenceNO: ABIIPMP42D4 4.40E−08 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFSTDTM 47GWFRQAPGKEREFVAAVTWNSGRTNYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRLVVGGTSVGDWRYWGQGTQVTVSS ABIIPMP111B4  5.1E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFWTDTM 48 GWFRQAPGKEREFVAAVTWSSGRANYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRG VVGGWVVVDWRYVVGQGTQVTVSS ABII111E5cI18.80E−09 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFLTDTM 49GWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGREVQDWRYWGQGTQVTVSS ABIIPMP11106 6.00E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFWTDTM 50 GWFRQAPGKEREFVAAVTWNSGRLNYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRQ VVGGVQVLDWRYWGQGTQVTVSS ABIIPMP111F23.40E−09 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFSTDTM 51GWFRQAPGKEREFVAAVTWNSGRTNYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTANRHSVGRLSVGDWRYWGQGSQVTVSS ABIIPMP111E4 6.10E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFSTDTM 52 GWFRQAPGKEREFVAAVTWNSGRTNYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAARLT VGSLSVGDWRYWGQGTQVTVSS ABIIPMP111C48.60E−09 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFLTDTM 53GWFRQAPGKEREFVAAVTWNSGRANYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRSVVGGVGVWDWRYWSQGTQVTVSS ABIIPMP111B5 4.40E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 54 GWFRQAPGKEREFVAAVTWNSGRNNYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRL VVGGGGVRDWRYWGQGTQVTVSS ABIIPMP111B91.90E−08 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFGTDTM 55GWFRQAPGKEREFVAAVTWNSGRANYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGGCVKDWRYWGQGTQVTVSS ABII111B5_ 5.30E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFKTDTM 56 R30KGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNMVEDWRYWGQGTQVTVSS ABII111B5_ 5.00E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFWTDTM 57 R30WGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNMVEDWRYWGQGTQVTVSS ABII111B5_ 9.20E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 58 N106TGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGTMVEDWRYWGQGTQVTVSS ABII111B5_ 1.00E−08 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 59 N106VGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGVMVEDWRYWGQGTQVTVSS ABII111B5_ 7.30E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 60 F101WGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRWVVGGNMVEDWRYWGQGTQVTVSS ABII111B5_ 4.30E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 61 M107EGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNEVEDWRYWGQGTQVTVSS ABII111B5_ 2.90E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 62 M107R =GWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF ABII002TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFV VGGNRVEDWRYWGQGTQVTVSS ABII111B5_4.70E−09 n.d. EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 63 E109WGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNMVWDWRYWGQGTQVTVSS ABII111B5_ 3.70E−09 n.d.EVQLVESGGGLVLAGGSLRLSCVHSGPTFRTDTM 64 E109QGWFRQAPGKEREFVAAVTWNSGRINYADSVKGRF TVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNMVQDWRYWGQGTQVTVSS ABII003 2.10E−09 n.d.EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM 65 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0042.10E−09 56 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 66GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII005 3.30E−09 48.2EVQLLESGGGLVLPGGSLRLSCAHSGPTFRTDTM  67 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0068.00E−08 51.7 EVQLLESGGGLVLPGGSLRLSCVASGPTFRTDTM  68GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII007 1.60E−09 53.2EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  69 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNTRNAAYLQMSGLKDEDTAVYYCTAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0081.90E−09 51.9 EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  70GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNSRNAAYLQMSGLKDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII009 1.70E−09 2.2EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  71 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTKNAAYLQMSGLKDEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0101.90E−09 54.8 EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  72GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNTAYLQMSGLKDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII011 2.40E−08 54.6EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  73 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNALYLQMSGLKDEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0121.50E−09 53.5 EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  74GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMNGLKDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII013 1.60E−09 52.2EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  75 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSSLKDEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0141.30E−09 52.3 EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  76GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMSGLRDEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII015 1.80E−09 55.5EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  77 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSGLKAEDTAVYYCTAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0162.40E−09 57.9 EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM  78GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMSGLKPEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII017 2.40E−09 59.4EVQLLESGGGLVLPGGSLRLSCVHSGPTFRTDTM 79 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCAAHRF VVGGNRVEDWRYWGQGTLVTVSS ABII0183.20E−09 0.1 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 80GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTVSRDNTRNAAYLQMSGLKPEDTAVYYCTAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII019 1.40E−08 71.6EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 81 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0201.10E−07 69.8 EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 82GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNTLYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII021 2.40E−08 69.9EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 83 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNAVYLQMNSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0221.60E−08 68.6 EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 84GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNAVYLQMSSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII023 5.80E−09 64.7EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 85 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNAAYLQMNSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0246.00E−09 63.6 EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 86GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNAAYLQMSSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII025 1.80E−08 70.3EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 87 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTVYLQMSSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0265.10E−09 66.8 EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 88GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII027 4.40E−09 65.3EVQLLESGGGLVQPGGSLRLSCAHSGPTFRTDTM 89 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMSSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0281.10E−08 73.8 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 90GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII029 8.90E−08 74.3EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 91 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0301.20E−08 73.7 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 92GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNAVYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII031 1.30E−08 72.1EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 93 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNAVYLQMSSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0324.90E−09 68.3 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 94GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNAAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII033 4.00E−09 66.2EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 95 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNAAYLQMSSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0341.00E−08 72.5 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 96GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNTVYLQMSSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII035 4.00E−09 68.7EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 97 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII0363.50E−09 67.5 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 98GWFRQAPGKGREFVAAVTWNSGRINYADSVKGR FTISRDNSKNTAYLQMSSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII037 3.10E−09 66.3EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTM 99 GWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRAEDTAVYYCAAHRFV VGGNRVEDWRYWGQGTLVTVSS ABII60A10 =1.90E−08 68.5 EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYNM 100 ABII050GWFRQAPGKEREFVAAVSRSGVSTYYADSVKGRF TISRDNAKNAVYLQMNSLKPEDTAIYYCGAAYRGTAINVRRSYDSWGQGTQVTVSS ABII051 1.80E−08 66.2EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM 101 GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNAKNAVYLQMNSLRPEDTAIYYCGAAYRG TAINVRRSYDSWGQGTLVTVSS ABII0521.90E−08 66.1 EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM 102GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNAVYLQMNSLRPEDTAIYYCGAAYRGTAINVRRSYDSWGQGTLVTVSS ABII053 2.00E−08 67.3EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM 103 GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAIYYCGAAYRG TAINVRRSYDSWGQGTLVTVSS ABII0545.10E−08 62 EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM 104GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNAKNALYLQMNSLRPEDTAIYYCGAAYRGTAINVRRSYDSWGQGTLVTVSS ABII055 2.20E−08 66.2EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  105GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNAKNAVYLQMNSLRPEDTAVYYCGAAYRGTAINVRRSYDSWGQGTLVTVSS ABII056 2.90E−08 69.8EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM 106 GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNAKNAVYLQMNSLRPEDTAIYYCAAAYRG TAINVRRSYDSWGQGTLVTVSS ABII0574.60E−08 71.5 EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  107GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYDSWGQGTLVTVSS ABII058 7.30E−08 68.9EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  108GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTLYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYDSWGQGTLVTVSS ABII059 3.30E−08 72.7EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  109GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS ABII060 5.00E−08 69.2EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  110GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTLYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS ABII061 2.40E−09 69.3EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNM  111GWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCGAAYRGTAINVRRSYSSWGQGTLVTVSS

EXAMPLES Example 1 Immunization of Llamas with A-Beta for the Inductionof Humoral Immune Responses

Generation of Monomeric A-Beta Peptide (BAM):

Monomeric A-beta peptide (BAM) is prepared via trifluoroacetic acid(TFA; Sigma)/1,1,1,3,3,3-hexafluor-2-propanol (HFIP; Fluka) treatment.The lyophilized A-beta peptide is dissolved in its original vial in 100%TFA to a final concentration of 1 mg/ml. The solution is then evaporatedin a speedvac at room temperature. After this and all subsequentevaporation steps the remaining pellet is placed on ice. The pellet isresuspended in HFIP and again evaporated in a speedvac at roomtemperature followed by another HFIP solubilization after which thesolution is divided into aliquots of appropriate volumes. The aliquotsare evaporated in a speedvac at room temperature and the pellets arestored at −80° C. Immediately before use, the TFA/HFIP treated A-betapeptide aliquot is dissolved in 100% DMSO via repeated up and downpipetting. The A-beta peptide solution is centrifuged at 14000 rpm for10 minutes to remove possible aggregates and the supernatant is used inthe different assays.

Immunization of Llamas:

Llamas are immunized with aggregated A-beta peptides (BAA) andoligomeric A-beta peptides (BAO) by injecting the immunogensintramuscularly in the neck area applying decreasing doses over time.BAA consist of a mixture of synthetic A-beta(1-40) and A-beta(1-42)peptides which are prepared essentially as described by Schenk et al.,1999, Nature 400: 173-177. BAO are prepared essentially as described byKayed et al. (2003), Science 300:486-489. The first two antigeninjections consist each time of 100 μg of antigen per llama, while forall following administrations, the dose is reduced to 50 μg per llama.Llamas 144 and 145 are vaccinated with freshly prepared BAA. In total,nine BAA antigen doses are injected as an emulsion using Freund'sComplete Adjuvant (first injection) or Stimune (all following immunogenboosts) in intervals of maximally 16 days. Llamas 129, 130, 177 and 178are vaccinated with BAO. In total, six to nine BAO antigen doses areinjected, as an emulsion using Freund's Complete (first injection) andFreund's Incomplete Adjuvant or Stimune (all following antigen boosts)in intervals of maximally 18 days. Llamas are also immunized with A-betapeptide fragment 1-16 conjugated to bovine serum albuming (BSA; llamas181 and 186) or A-beta peptide fragment 1-30 conjugated to BSA (llamas185 and 187). Four antigen injections are administered in 14-dayintervals, using Freund's Complete or Freund's Incomplete adjuvant withantigen doses decreasing from 100 μg (first two injections) to 50 μg(two following injections) per llama. Immediately before the start ofeach immunization scheme a pre-immune serum sample and at regular timepoints during the immunization experiment multiple immune serum samplesare collected to evaluate the A-beta peptide specific humoral responseof the distinct animals.

To monitor the A-beta peptide specific serum titers via ELISA, 2 μg/mlA-beta(1-40), biotinylated at the C-terminus (Anaspec), is immobilisedfor two hours at room temperature on a Neutravidin-coated (0.2-0.5 μgper well) 96-well Maxisorp plate (Nunc). Wells are blocked with a caseinsolution (1% in PBS). After addition of a dilution series of pre-immuneand immune serum samples, specifically bound llama immunoglobulins aredetected using a goat anti-llama horseradish peroxidase conjugate(Bethyl Lab. Inc.), allowing to detect the humoral response mediated byboth the conventional and heavy-chain only antibodies. In certain cases,a consecutive ELISA is performed to evaluate specifically theheavy-chain antibody mediated response via detection with mouse mAbsspecifically recognizing the heavy-chain only llama IgG2 and IgG3antibodies (Daley et al., 2005, Clin. Diagn. Lab. 1 mm. 12:380-386),followed by a rabbit anti-mouse-HRP conjugate (DAKO). ELISAs aredeveloped using TMB (Promega) as the chromogenic substrate andabsorbance is measured at 450 nm. For all four immunogen formats (BAA,BAO, A-beta(1-16)-BSA and A-beta(1-30)-BSA) both conventional andheavy-chain antibody mediated immune responses specific to(biotinylated) monomeric A-beta(1-40) are detected. Serum samples thatare positive against monomeric A-beta(1-40) are also positive whentested against BAA directly coated onto a Maxisorp plate, suggesting thepresence of at least partially common epitopes in the monomeric A-betaand BAA preparations used.

Example 2 Isolation of A-Beta Binding VHH Domains (VHHs) from ImmunizedLlamas

Cloning of the Heavy-Chain Only Antibody Fragment Repertoires:

Following the final immunogen injection, immune tissues as the source ofthe B-cells producing the heavy-chain antibodies are collected from theimmunized llamas. Typically, two 150-ml blood samples, are collected 4and 8 days after the last antigen injection and one lymph node biopsy,collected 4 days after the last antigen injection are collected peranimal. From the blood samples, peripheral blood mononuclear cells(PBMCs) are prepared using Ficoll-Hypaque according to themanufacturer's instructions (Amersham Biosciences). From the PBMCs andthe lymph node biopsy, total RNA is extracted, which are used asstarting material for RT-PCR to amplify the VHH encoding gene segments(formerly described in WO2005/044858). For each immunized llama, alibrary is constructed by pooling the total RNA isolated from allcollected immune tissues of that animal. In short, the PCR-amplified VHHrepertoire is cloned via specific restriction sites into a vectordesigned to facilitate phage display of the VHH library. The vector isderived from pUC119 and contained the LacZ promoter, a M13 phage gillprotein coding sequence, a resistance gene for ampicillin orcarbenicillin, a multiple cloning site and a hybrid gIII-pelB leadersequence (pAX050). In frame with the VHH coding sequence, the vectorencodes for a C-terminal c-myc tag and a (His)₆ tag. Phage are preparedaccording to standard protocols and are stored after filtersterilization at 4° C. for further use.

Selection of A-Beta Specific VHHs Via Phage Display:

VHH repertoires obtained from all llamas and cloned as phage library areused in different selection strategies applying a multiplicity ofselection conditions. Variables include:

i) the A-beta peptide format (A-beta(1-40) C- or N-terminallybiotinylated, A-beta(1-16) C-terminally biotinylated, A-beta(1-30)N-terminally biotinylated and glutathione-S-transferase (GST) fusionssuch as A-beta(1-42)-GST, A-beta(1-10)-GST or GST-A-beta(1-42)),ii) the A-beta peptide aggregation status (monomeric A-beta, BAO orBAA),iii) the antigen presentation method (solid phase: directly coated orvia a biotin-tag onto Neutravidin-coated plates; solution phase:incubation in solution followed by capturing on Neutravidin-coatedplates),iv) the antigen concentration andv) different elution methods (trypsin or TEA).

Selections are performed as follows: antigen preparations for solid andsolution phase selection formats are presented as described above atmultiple concentrations. After 2 h incubation with the phage librariesfollowed by extensive washing, bound phage are eluted with trypsin (1mg/ml) or TEA for 15 minutes. In case trypsin is used for phage elution,the protease activity is immediately neutralized applying 0.8 mMprotease inhibitor ABSF. As control, selections w/o antigen areperformed in parallel. Phage outputs that show enrichment overbackground (non-antigen control) are used to infect E. coli. Infected E.coli cells are either used to prepare phage for the next selection round(phage rescue) or are plated on agar plates (LB+Amp+2% glucose) foranalysis of individual VHH clones. In order to screen a selection outputfor specific binders, single colonies are picked from the agar platesand grown in 1-ml 96-deep-well plates. The placZ controlled VHHexpression is induced by adding IPTG (0.1-1 mM final) in absence ofglucose. Periplasmic extracts (in a volume of ˜80 μl) are preparedaccording to standard methods.

Screening of Periplasmic Extracts for Binding to A-Beta:

2 μg/ml A-beta(1-40), biotinylated at the C-terminus (Anaspec), iscaptured for two hours at room temperature on a Neutravidin-coated(0.2-0.5 μg per well) 96-well Maxisorp plate (Nunc). Wells are blockedwith a casein solution (1% in PBS). After addition of typically a10-fold dilution of the periplasmic extracts, VHH binding is detectedusing a mouse anti-myc and an anti-mouse-HRP conjugate (DAKO). Clonesthat give an ELISA signal of minimally two-fold above background areretained for sequence analysis. VHHs that are able to bind(biotinylated) monomeric A-beta(1-40) can be allocated to 16 differentB-cell lineages. Clones derived from the same B-cell lineage share ahighly similar CDR3 sequence and are thus likely to recognize the sameepitope. Table VIII summarizes the selection parameters that leads tothe identification of a representative VHH of each of the 16 B-celllineages. In Table IX, the amino acid sequences of the VHHs listed inTable VIII are shown.

TABLE VIII Selection parameters are used for the identification ofA-beta specific VHH B-cell lineages Selection format (immobilized orPhage Rounds of VHH ID Library captured A-beta peptide concentration)elution selection ABII1E11 130 BAA (200 ng) trypsin 2 ABII5D2 145 BAA(200 ng) trypsin 1 ABII14D4 130 BAA (200-40 ng) trypsin 2 ABII35C7 Poolof 144, A-beta(1-42)GST and GST-A-beta(1-42) TEA 2 145, 129, 130 (10μg/well) ABII35D2 Pool of 144, A-beta(1-42)-GST and GST-A-beta(1-42) TEA2 145, 129, 130 (10 μg/well) ABII35G2 Pool of 144, A-beta(1-42)GST andGST-A-beta(1-42) TEA 2 145, 129, 130 (10 μg/well) ABII42D4 178biotinylated A-beta(1-40) (100 nM) trypsin 2 ABII42B10 178 biotinylatedA-beta(1-40) (10 nM) trypsin 2 ABII42F5 178 biotinylated A-beta(1-40)(100 nM) trypsin 2 ABII42E10 178 biotinylated A-beta(1-40) (10 nM)trypsin 2 ABII42G10 178 biotinylated A-beta(1-40) (10 nM) trypsin 2ABII60A10 185 Round I: BAA (200 ng) trypsin 2 Round II: biotinylatedA-beta(1-40) (1 nM) ABII60D2 181 Round I: BAA (200 ng) trypsin 2 RoundII: biotinylated A-beta(1-40) (10 nM) ABII60G11 185 Round I: BAA (200ng) trypsin 2 Round II: biotinylated A-beta(1-40) (1 nM) ABII60H5 181Round I: BAA (200 ng) trypsin 2 Round II: biotinylated A-beta(1-40) (1nM) ABII61F6 187 Round I: BAA (200 ng) trypsin 2 Round II: biotinylatedA-beta(1-40) (10 nM)

TABLE IX  Amino acid sequencs of resulting representative VHHs: SEQVHH ID Wild type monovalent A-beta binding VHHs ID NO: ABII1E11EVQLVESGGGLVQPGGSLRLSCAASGRTFSDYNMGWFHQAPGKEREIVA 112AISRSGRSTYYTVSVEGRFTISRDNAKNTVDLEMNSLKPEDTGIYYCAANSAGRAINLPLYKYWGQGTQVTVSS ABII14D4EVQLVESGGGLVQAGGSLRLSCAASGRTFSTYNMAWFRHAPGKEREFVA 113AISRSGGSTYYVDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAAPRGRSIVTTATYDYWGQGTQVTVSS ABII5D2EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWVRQAPGKERELVA 114TISQSGGLRSYADSVKGRFTISRDNAKNTVYLQMNSLTPGDTAVYYCAAQARATAWSPQRVDYWGQGTQVTVSS ABII35C7EVQLVESGGGLVQPGGSLRLSCAASGRTFSIYNMGWFRQAPGKEREFVA 115AISRSGSSTYYGDSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYHCAAARFGTPINTRGSYDYWGQGTQVTVSS ABII35D2EVQLVESGGGLVQAGGSLRLSCVASGLTFSSYNMGWFRQAPGKEREFVA 116AISRSGGSTYYTDSVKGRFTISRDSSKNTVYLQMNSLKPEDTADYYCAAALFGSAINLLSEYRYWGQGTQVTVSS ABII35G2EVQLVESGGGLVQAGGSLRLSCVASGRTFSNYGMGWFRQAPGKDREFV 117AAISRSGGTTYYEDDVKGRFTISRDNAKNSVYLQMNSLKPEDTAVYYCAARPSYVAVNIAASYNNWGQGTQVTVSS ABII42D4EVQLVESGGGLVLAGGSLRLSCVHSGPTFSTDTMGWFRQAPGKEREFVA 118AVTWNSGRTNYADSVKGRFTVSRDNTRNAAYLQMSGLKDEDTAVYYCTAHRLVVGGTSVGDWRYWGQGTQVTVSS ABII42B10EVQLVESGGGLVQRGGSLRLSCAASGRTFSNLNMGWFRQAPGKEREFQ 119AAISRSGGTTYYADSVKGRFTISRDNAKSTVFLQMNSLKPEDTAVYYCAAASPGGPINYGRAYDSWGQGTQVTVSS ABII42F5EVQLVESGGGLVQAGDSLRLSCTASGRTFTDYNIGWFCQAPGKEREFVAA 120ISGSGGSTYYADSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYACAAAQRRLAVNVDTSYNVWGQGTQVTVSS ABII42E10EVQLVESGGGLVQPGGSLRLSCAASGLTFTLYTMGWFRQAPGKEREFVA 121AISASGGTTYYADSVKGRFALSRDNAKNTVFLQMNTLKPEDTAEYLCAAAFRGFAINTPTSYNYWGQGTQVTVSS ABII42G10EVQLVESGGGLVQAGGSLRLSCLFSGRTFSTNGVGWFRQVPGKEREFVS 122AINWSGSKTNYAEPVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAAYRTSISRYEYAYWGQGTQVTVSS ABII60A10EVQLVESGGGLVQAGGSLRLSCAASGRTFNNYNMGWFRQAPGKEREFV 123AAVSRSGVSTYYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAIYYCGAAYRGTAINVRRSYDSWGQGTQVTVSS ABII60G11EVQLVESGGGSEQAGGSLRLSCATSGRAFSVYAWFRQAPGKERTFVAAV 124AWVGGSTFYSDSVKGRFTISRDNAKNTVFLHMNSLKPEDTAAYYCAARLYGGRWYNSPRVDDFEYWGQGTQVTVSS ABII60D2EVQLVESGGGSVQAGGSLRLSCAYSGSIFSIKTMGWYRQAPGKQRELVG 125RITSGDSTNYADSVKGRFTISRDKAKTTVYLQMNNLKPEDTAVYYCAARRP WPRSDVWGQGTQVTVSSABII60H5 EVQLVESGGGLVQVGGSLRLSCAASGNIGSINAMGWYRQAPGKEREWVA 126IITNSGSVNYGPDSVKGRFTISGDNAKNRVYLQMDSLKPEDTAVYYCAAES WGRSPLKYLGQGTQVTVSSABII61F6  EVQLVESGGGLVQTGGSLRLSCAASGSTVNINAMGWYRQAPGKQRELVAI 127INKRGVTNYADSTEGRFTISRDNSKRTLYLQMNSLKPEDTAVYYCNAVVGR YGRTYGYWGQGTQVTVSSThe total number of variants (minimally 1 amino acid difference) foundfor each B-cell lineage is 1 (ABII5D2), 195 (ABII42D4), 1 (ABII42G10), 1(ABII60D2), 1 (ABII60H5), 23 (ABII1E11), 3 (ABII14D4), 3 (ABII35C7), 6(ABII35G2), 1 (ABII35D2), 7 (ABII42B10), 2 (ABII42F5), 3 (ABII42E10), 2(ABII60A10), 2 (ABII60G11) and 15 (ABII61F6).

To identify the B-cell lineage variant with the best binding properties,periplasmic extracts of all VHH variants were used to determine theoff-rate (Biacore T100, GE Healthcare). Monomeric A-beta(1-40)(biotinylated at the C-terminus; Anaspec), monomeric A-beta(1-16)(biotinylated at the C-terminus; Bachem) and monomeric A-beta(12-28)(biotinylated at the N-terminus; Bachem), is prepared as described inExample 1, are irreversibly captured via streptavidin on three differentchannels of the same SA sensor chip (GE Healthcare). Surfaces are firstwashed via 3×1-minute injections of surface wash buffer (1M NaCl in 50mM NaOH) followed by injecting biotinylated A-beta at 50 nM up to atarget level of 100 RU. After capturing, surfaces are blocked byinjecting an excess (200 μg/ml) of d-biotin for 180 s at 5 μl/min. Areference surface is washed and blocked with d-biotin. HBS-EP+buffer isused as the running buffer and experiments are performed at 25° C. Foroff-rate screening, periplasmic extracts of VHHs are injected at a10-fold dilution for 2 min at 45 μl/min and are allowed to dissociatefor 10 min. Purified reference binders (VHHs at 100 nM, 3D6 Fab at 10nM) are injected as positive control samples and evaluated at least atthe beginning and at the end of each experiment. Between different VHHsamples, the surfaces are regenerated with regeneration buffer (50 mMNaOH) for 25 s at 45 μl/min followed by 10 s 6M GuHCl at 45 μl/min ifregeneration is incomplete. Off-rates are calculated from thesensorgrams obtained from the channel with captured biotinylatedA-beta(1-40). The variants of only two VHH B-cell lineages, ABII42D4 andABII60G11, showed a monophasic binding pattern and allowed thecalculation of off-rates via a 1:1 interaction model: 6.1E-3 s⁻¹ and2.7E-2 s⁻¹, respectively. For all other VHH B-cell lineages, 2 differentsections of the dissociation curve are fitted separately, rendering ak_(d1) (calculated from the dissociation frame between 125 and 160 s)and a k_(d2) (400 s-700 s). Data are double referenced by subtraction ofthe curves on the reference channel and of a blank running bufferinjection. Sensorgrams are evaluated by fitting a 1:1 dissociation modelusing the Biacore T100 Evaluation software v1.1.1. Although binding ofVHHs ABII60D2 and ABII60H5 to A-beta is observed in the screening ELISA,these VHHs show poor binding to the sensor chip.

For studying binding characteristics of monovalent non-VHH binders, Fabfragments of monoclonal antibodies m266 and 3D6 are used. Monoclonalantibody 3D6 is described in Johnson-Wood et al., 1997, Proc. Natl.Acad. Sci. 94:1550-1555 and in Bard et al., 2000, Nature Medicine6:916-919 and specifically binds to amino acid residues 1 to 5 of A-beta(N-terminal epitope). Monoclonal antibody m266 is described in Seubertet al., 1992, Nature 359:325-327 and specifically binds to amino acidresidues 16 to 24 of A-beta (central epitope of A-beta). Fab fragments,comprising the variable light chain (V_(L)), variable heavy chain(V_(H)), constant light chain (C_(L)) and constant domain 1 of the heavychain (CH₁) of the respective antibody (sequences as given below) and,additionally, a C-terminal c-myc tag and a hexa-histidine ((His)₆ tag)are cloned, expressed and purified according to conventional techniques,using E. coli as the host organism and immobilized metal affinitychromatography (IMAC) and size exclusion chromatography (SEC) forpurification. Purified Fab fragments of m266 and 3D6 show, in theabove-described binding experiment, off-rates of 4.0E-5 s⁻¹ and 4.7E-4s⁻¹, respectively.

VL- and VH-sequences of Fab fragment of 3D6: VL: (SEQ ID NO: 128)YVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRIEAEDLGLYYCWQGTH FPRTFGGGTKLEIK VH:(SEQ ID NO: 129) EVKLVESGGGLVKPGASLKLSCAASGFTFSNYGMSWVRQNSDKRLEWVASIRSGGGRTYYSDNVKGRFTISRENAKNTLYLQMSSLKSEDTALYYCVR YDHYSGSSDYWGQGTTVTVSSVL- and VH-sequences of Fab fragment of m266: VL: (SEQ ID NO: 130)DVVMTQTPLSLPVSLGDQASISCRSSQSLIYSDGNAYLHWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVETEDLGVYFCSQSTH VPWTFGGGTKLEIK VH:(SEQ ID NO: 131) EVKLVESGGGLVQPGGSLKLSCAVSGFTFSRYSMSWVRQTPEKRLELVAQINSVGNSTYYPDTVKGRFTISRDNAEYTLSLQMSGLRSDDTATYYCAS GDYWGQGTTLTVSS

Example 3 Characterization of Purified VHHs

The VHH variants with the slowest dissociation rates for each B-celllineage are recloned into an expression vector derived from pUC119,which contains the LacZ promoter, a resistance gene for eitherampicillin or kanamycin, a multiple cloning site and a hybrid gIII-pelBleader sequence. In frame with the VHH coding sequence, the vectorencodes for a C-terminal c-myc tag and a (His)₆ tag. VHHs are producedin E. coliG1 and are purified via immobilized metal affinitychromatography (IMAC) and size exclusion chromatography (SEC) resultingin 95% purity as assessed via SDS-PAGE.

3.1 VHH Binding to Immobilized A-beta (ELISA):

To quantify the binding of the VHHs to monomeric A-beta peptide (BAM),VHHs are applied as dilution series in an ELISA using the same setup asdescribed in Example 2. Except for VHHs ABII60D2 and ABII60H5, EC50values can be calculated and are summarized in Table X. The most potentVHHs interacting with the N-terminal epitope of A-beta (amino acids 1 to16) or the central epitope of A-beta (amino acids 12 to 28) aredetermined to be ABII42D4 (EC50 of 14.2 nM) and ABII60A10 (EC50 of 4.9nM), respectively.

VHHs that give detectable signals against BAM are tested for binding toA-beta peptide aggregates (BAA) in ELISA, applying a similar set-up asdescribed in Example 2. Antigen is prepared and immobilized as describedin Bohrmann et al., 1999, J Biol Chem 274: 15990-15995. In short, 100 μlof 10 μg/ml of A-beta(1-40) diluted in TBS buffer (50 mM Tris, 150 mMNaCl pH 7.4 and 0.05% Na-azide) from a DMSO stock solution of 2 mg/mlsynthetic A-beta(1-40) (Bachem) is allowed to aggregate for 60 h at 37°C. All VHHs that recognize BAM also recognize BAA and the respectiveEC50 values are summarized in Table X.

TABLE X EC50 values for purified VHHs in BAM and BAA ELISA VHH B-cellBAM ELISA BAA ELISA TR-FRET lineage Epitope EC50 in nM EC50 in nM IC50in nM ABII1E11 12-28 27.3 19.2  844* ABII5D2  1-16 54.1 11.7 >1000   ABII14D4 12-28 124 6.8 744 ABII35C7 12-28 1.9 1.3  206* ABII35G2 12-2810.7 5.4  645* ABII35D2 12-28 37.5 12.8  773* ABII42D4  1-16 14.2 0.9  46.8 ABII42B10 12-28 10.8 2.1   85.6 ABII42F5 12-28 41.1 20.0  228*ABII42E10 12-28 17.5 2.6  205* ABII42G10  1-16 87.0 49.5 >1000   ABII60A10 12-28 4.9 0.8   11.1 ABII60G11 12-28 187 6.0   34.4 ABII60D2 1-16 >1 μM ND ND ABII60H5  1-16 >1 μM ND ND ABII61F6 12-28 469 14.5 967* 2D2 12-28 15.8 4.7  404* 2G6  1-16 98.6 7.2 >1000    Fab266 16-24* 1.8 1.8    0.52 Fab3D6  1-5* 1.8 0.3    3.4 ND: not determined;*IC50 calculated from extrapolated curves

3.2 VHH Binding to A-Beta in Solution (TR-FRET):

Interaction of anti-A-beta VHHs and A-beta peptide in solution areevaluated using a Time Resolved Fluorescence Resonance Energy Transfer(TR-FRET) competition assay. In two different setups, competition witheither the A-beta(1-40) peptide—ABII42D4 interaction (N-terminal regionspecific) or with the A-beta(1-40)—ABII60A10 interaction (central regionspecific) are tested. For the assay monomeric A-beta(1-40) (biotinylatedat the C-terminus; Anaspec) labeled with streptavidin-Europium chelateand VHHs ABII42D4 or ABII60A10 labeled with AlexaFluor647 are incubatedfor 1 h with different concentrations of a non-labeled competitor (VHH,IgG or Fab). The labeled compounds are used at concentrations of 0.2 nM(A-beta(1-40)), 50 nM (ABII42D4) and 10 nM (ABII60A10), respectively andthe fluorescence signal emitted upon binding interaction is detected at665 nm. IC50 values are determined in the ABII42D4 and ABII60A10 TR-FRETassays resulting in the following potency ranking for N-terminal regionspecific binders tested: monoclonal antibody 3D6 (2.5 nM)>Fab fragment3D6 (3.4 nM)>ABII42D4 (46.8 nM)>ABII5D2=42G10 (>1000 nM). The mostpotent central-region specific VHH identified via this assay wasABII60A10, shows an IC50 of 11.1 nM while benchmark antibodies gaveIC50s of 0.50 nM (monoclonal antibody m266) and 0.52 nM (Fab fragment ofm266).

For comparative reasons, anti-A-beta VHHs as disclosed previously (the“reference VHHs”) are generated and purified as described above, usingthe sequence information as available from the international patentpublications indicated below:

VHHs 2D2 and 2G6: WO2006/40153;

VHHs 3A, 1B, 11G, 4D, and 8B: WO2007/35092; and

VHH 31-1: WO2004/44204.

Amino acid sequences thereof are shown in Table XI.

TABLE XI  Amino acid sequences of reference VHHs VHH SEQ IDA-beta binding reference VHHs ID NO: 2D2AVQLVDSGGGLVQAGGSLRLSCAVSGGTFSSIGMGWFRQAP 132GKEREFVGAISRSGDSTYYADSVKGRFTISRDGAKNTVYLQMNSLKDEDTAVYYCAGRPAGTAINIRRSYNYWGQGTQVTVSS 2G6QVKLEESGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRQAP 133GKGLEWVSTISPRAANTYYADSVKGRFTISRDNAKNTLYLQMNSLEPDDTALYYCAKSLRYRDRPQSSDFLFWRQGTQVTVSS 3AAVQLVESGGGLVRDGDSLRLSCAASGRTFSSYVMGWFRQAP 134GKEREFVAAIGWSGGSTAYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAVYYCASAPTRWVPRDSRFYDRWGQGTRVTVSS 1BQVQLQESGGGLVQPGGSLRLSCAASEFTLDYYSIAWFRQAPG 135KEREGVSCISSYDGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAGIRDWATLREYEYDDWGQGTQVTVSS 11GQVQLQESGGGLVQPGGSLRLSCAASGSIFSINTMAWYRQAPG 136KERDLVAAIISSGSTNYADSVKGRFTISRDNTKNTVYLQMNSLKLEDTAVYYCNAAIRRSVIDAWGAYWGQGTQVTVSS 4DQVQLQESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAP 137GKEREFVATIRWNGDYADSVRDRFTISRDDAKNTVFLQMNSLKPEDTAIYYCAARLGPRTSQAALYRYWGQGTQVTVSS 8BAVQLVESGGGLVQAGGSLRLSCAASGRTFSSYVMGWFRQAP 138GKEREFVAAIGWSGGSTAYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAVYYCASAPTRWVPRDSRFYDRWGQGTRVTVSS 31-1AEVQLQASGGGSVQPGGSLRLSCAASGFIFGWSTMSWVRQA 139PGKGLEWVSTISGGGSATTYTDSVKGRFTISRDRAKNTLYLQMNSLKPEDTAIYYCNADVSTGFRYQRKDYWGRGTQVTVSS

Compared to the reference VHHs 2G6 (N-terminal region specific) and 2D2(central-region specific), VHHs 42D4 and 60A10 show 21fold and 36foldimproved IC50s, respectively (cf. Table X).

3.3 Determination of the Affinity of A-Beta Peptide-VHH Interaction:

Affinities for the A-beta peptide-VHH/Fab interaction are determined viasurface plasmon resonance (Biacore) using C-terminally biotinylatedA-beta(1-40) captured on a streptavidin sensor chip as described before(Example 2). Purified VHHs or Fab fragments are injected at 5 differentconcentrations (between 2.5 and 300 nM for ABII42D4 and ABII60A10) for 2min and allowed to dissociate for 10 min. For ABII42D4 but not forABII60A10 association and dissociation curves can be fitted using a 1:1interaction model (Langmuir binding) and an accurate K_(D) value can bedetermined. The affinities are found to be 16 nM for VHH ABII42D4, 5.1nM for the Fab fragment of 3D6, and 0.3 nM for the Fab fragment of m266,respectively.

3.4 Epitope Mapping Via Surface Plasmon Resonance:

Binding specificity of the VHHs are determined via surface plasmonresonance (Biacore) using A-beta(1-16) (biotinylated at the C-terminus;Bachem) and A-beta(12-28) (biotinylated at the N-terminus; Bachem)peptides are captured on a streptavidin sensor chip as described before.Five of the VHHs listed in Table IX above are found to interact with theN-terminal region of the Abeta peptide (ABII5D2, ABII42D4, ABII42G10,ABII60D2 and ABII60H5), and 11 VHHs are found to interact with thecentral region (ABII1E11, ABII14D4, ABII35C7, ABII35G2, ABII35D2,ABII42B10, ABII42F5, ABII42E10, ABII60A10, ABII60G11 and ABII61F6; cf.Table X).

3.5 In Vitro A-Beta Aggregation Assay:

VHHs are tested in an in vitro A-beta aggregation assay to assesswhether aggregation can be inhibited or reduced. In vitro A-betaaggregation is measured using Thioflavin T (ThT) fluorescence, whichundergoes a typical red shift upon A-beta fibril formation (Levine, H.1993. Protein Science (2): 404-410). For the assay, stock solutions ofsynthetic A-beta(1-40) (Bachem) in 100% DMSO and ThT in 25 mMglycine-NaOH pH8.5 is prepared and stored at −20° C. Before usage, theA-beta(1-40) stock solution is diluted to 56 μM in aggregation buffer(50 mM sodium phosphate, 100 mM NaCl pH5, 0.02% NaN3). Fab fragments andVHHs are tested as dilution series in D-PBS using concentrations between56 and 7 μM. Equal volumes (20 μl) of the 56 μM A-beta(1-40) solutionand the antibody/VHH samples or appropriate negative control are mixed(in triplicate) and mixtures are incubated in low adhesionmicrocentrifuge tubes for 48 hours at 37° C. in a dark environment.After transfer of 30 μl of the incubated samples into a black 96-wellflat bottom polypropylene plate (Greiner Bio-One), 250 μl of 2.5 μM ThT(stock solution diluted into 25 mM glycine-NaOH) solution are added andthe fluorescence signal is measured (Envision, PerkinElmer). The maximumfluorescence signal of A-beta(1-40) measured in the absence ofcompetitor is set as 100% aggregation (or 0% inhibition). The maximuminhibition at the highest VHH concentration tested (28 μM) is calculatedas an average of at least two independent experiments. While aconsistent background inhibition of approximately 25% is detected for anon-related (non-A-beta specific) VHH, monovalent Fab fragments 3D6 and266 show a reproducible dose-dependent inhibition with maximalinhibition of 79 and 85%, respectively. Out of the panel of VHHs tested,14D4, 35C7, 35G2, 35D2, 42B10, 42F5, 42E10 and 60A10 show a consistentinhibition of 78, 82, 76, 73, 77, 79, 75 and 80% of peptide aggregation,respectively (higher than the 25% background inhibition induced by thenon-specific control VHH).

Example 4 Affinity Maturation of VHHs

VHH ABII42D4 is subjected to two cycles of affinity maturation. In afirst cycle, individual CDR residues are mutated to all other 19 aminoacids. The following residues are targeted: CDR1: G26-G35; CDR2:V51-N58; and CDR3: H95-Y102 (numbering according to Kabat). Mutagenesisis performed in a PCR-based approach using degenerate oligonucleotidescontaining a NNS codon at the mutated position. PCR products are pooledfor each CDR and inserted via unique restriction sites into the ABII42D4gene template. Individual mutants are produced as recombinant proteinusing an expression vector derived from pUC119, which contain the LacZpromoter, a resistance gene for kanamycin, a multiple cloning site andan ompA leader sequence (pAX100). E. coli TG1 cells are transformed withthe expression vector library and plated on agar plates (LB+Amp+2%glucose). Single colonies are picked from the agar plates and grown in1-ml 96-deep-well plates. VHH expression is induced by adding IPTG (1mM). Periplasmic extracts (in a volume of ˜80 μl) are prepared accordingto standard methods and screened for binding to A-beta(1-40) in ELISAand in a Biacore off-rate assay as described before (Example 2).Mutations at six positions (S30, T57, L97, T100b, S100c, G100e) resultin slightly (˜2fold) improved off-rates.

In a second cycle, a combinatorial library is created by simultaneouslyrandomizing the six susceptible positions identified in cycle one. Forthis, the full length ABII42D4 gene is synthesized by overlap PCR usingoligonucleotides degenerated (NNS) at the randomization positions and arescue PCR is performed. The randomised ABII42D4 genes are inserted intoa phage display vector (pAX50) yielding a functional library size of6×10E7. Phages are prepared according to standard protocols. The phagelibrary is subjected to three rounds of solution phase selection against(biotinylated) monomeric A-beta(1-40) using streptavidin-coated magneticbeads (Dynal) for the capturing step. The antigen concentration isdecreased 10fold at each round starting from an antigen concentration of50 nM at round one. Bound phages are eluted with trypsin (1 mg/ml) for30 minutes and phage outputs are infected into E. coli TG1 forpreparation of periplasmic extracts of individual VHH clones. Screeningfor binding to A-beta(1-40) in ELISA and in a Biacore off-rate assay(Example 2) identifies clones with up to 10fold improved off-rates. Thebest ABII42D4 variants are cloned into expression vector pAX100 in framewith a C-terminal c-myc tag and a (His)6 tag. VHHs are produced in E.coli as His6-tagged proteins and purified by immobilized metal affinitychromatography (IMAC) and size exclusion chromatography (SEC). Theaffinities of the purified VHHs are determined via surface Plasmonresonance (Biacore) and IC50 values are determined in the ABII42D4TR-FRET competition assay (Example 3.2).

In an attempt to further improve the binding affinity, variantABIIPMP111B5 is used as template and divergent mutations found in clonesABIIPMP111E4 and ABIIPMP111B4 are introduced one by one. The resultingvariants are produced in E. coli and characterized as described before.ABII111B5_M107R, ABII111B5_M107E and ABII111B5_E109Q are identified asthe best variants with both, IC50 and K_(D), being more than 10foldimproved over original ABII42D4. Sequence information and biologicaldata for the variants mentioned above are summarized in Table VII above(first line: 42D4; lines 2 to 9: useful clones resulting from first twocycles of affinity maturation; lines 10 to 18: useful clones resultingfrom additional targeted mutations).

Example 5 Humanization of VHHs

The amino acid sequences of the anti-A-beta VHH ABII111B5_M107R(=ABII002; SEQ ID NO: 62) and of the anti-A-beta VHH ABII60A10(=ABII050; SEQ ID NO:100) are blasted against the human germline V_(H)sequence database. The human germline VH3-23 sequence (DP47; SEQ ID NO:140) in combination with JH5 showed the highest sequence identity toboth VHH sequences.

Sequence DP-47/VH3-23: (SEQ ID NO: 140)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK Sequence JH5:(SEQ ID NO: 153) NWFDSWGQGTLVTVSS

17 and 10 amino acid residues of ABII002 and ABII050, respectively, weresubstituted for humanization purposes creating a number of differentvariants of each VHH. All variants were assembled from oligonucleotidesusing a PCR overlap extension method, cloned into an expression vector(pAX100) and produced in E. coli. The sequence information and bindingdata for the obtained variants of ABII002, i.e. ABII003 to ABII037, andfor the obtained variants of ABII0050, i.e. ABII051 to ABII061, aresummarized in Table VII above (lines 19 to 53 and lines 55 to the lastline 65, respectively). Table XII additionally lists the degree ofhumanization of ABII002 to ABII036 and ABII050 to ABII060.

TABLE XII Humanized variants of ABII002 and ABII050 Clone % human- T_(m)Mean IC50 in name ization (° C.) TR-FRET (M) ABII002 77.8 57.5 2.0E−09ABII003 81.8 51.1 2.1E−09 ABII004 82.8 56.0 2.1E−09 ABII005 82.8 48.23.3E−09 ABII006 82.8 51.7 8.0E−08 ABII007 82.8 53.2 1.6E−09 ABII008 82.851.9 1.9E−09 ABII009 82.8 52.2 1.7E−09 ABII010 82.8 54.8 1.9E−09 ABII01182.8 54.6 2.4E−08 ABII012 82.8 53.5 1.5E−09 ABII013 82.8 52.2 1.6E−09ABII014 82.8 52.3 1.3E−09 ABII015 82.8 55.5 1.8E−09 ABII016 81.8 57.92.4E−09 ABII017 82.8 59.4 2.4E−09 ABII018 82.8 60.1 3.2E−09 ABII019 91.971.6 1.4E−08 ABII020 92.9 69.8 1.1E−07 ABII021 90.9 69.9 2.4E−08 ABII02289.9 68.6 1.6E−08 ABII023 90.9 64.7 5.8E−09 ABII024 89.9 63.6 6.0E−09ABII025 90.9 70.3 1.8E−08 ABII026 91.9 66.8 5.1E−09 ABII027 90.9 65.34.4E−09 ABII028 90.9 73.8 1.1E−08 ABII029 91.9 74.3 8.9E−08 ABII030 89.973.7 1.2E−08 ABII031 88.9 72.1 1.3E−08 ABII032 89.9 68.3 4.9E−09 ABII03388.9 66.2 4.0E−09 ABII034 89.9 72.5 1.0E−08 ABII035 90.9 68.7 4.0E−09ABII036 89.9 67.5 3.5E−09 ABII050 83.8 68.5 1.9E−08 ABII051 88.9 66.21.8E−08 ABII052 89.9 66.1 1.9E−08 ABII053 89.9 67.3 2.0E−08 ABII054 89.962.0 5.1E−08 ABII055 89.9 66.2 2.2E−08 ABII056 89.9 69.8 2.9E−08 ABII05792.9 71.5 4.6E−08 ABII058 93.9 68.9 7.3E−08 ABII059 92.9 72.7 3.3E−08ABII060 93.9 69.2 5.0E−08

According to the biological data summarized in Tables VII and XII above,all of the VHH variants listed therein can—at least as intermediates—beused for the construction of the polypeptides of the invention.Specifically, for use in humans, humanized VHH variants ABII003 toABII037 and ABII051 to ABII061 will be preferred. Particularly usefulvariants, showing a high degree of humanization but preserving theirbinding properties are ABII035 derived from the original clone 42D4, andABII059 derived from original clone 60A10. ABII035 binds to theN-terminal epitope of A-beta and comprises CDR sequences SEQ ID NOs:14to 16. ABII059 binds to the central epitope and comprises CDR sequencesSEQ ID NOs:17 to 19.

Example 6 Comparison of the Humanized VHHs to the Reference VHHs

Purified anti-A-beta reference VHHs (Example 3.2, Table XI) are testedfor binding as described in Example 3 to a SA sensor chip coated withC-terminally biotinylated monomeric A-beta(1-40). While ABII035 andABII059 showed RU levels >50 at injected concentrations of 11 and 3 nM,respectively, none of the VHHs 1B, 11G, 8B, 4D, 3A and VHH31-1 showbinding (>10 RUs) to the sensor chip at a concentration of 1000 nM underthe conditions tested. In the same experiment, reference VHHs 2G6 and2D2 show RU levels >50 at injected concentrations of 1000 and 100 nM,respectively, thus being much less potent than ABII035 and ABII059. Noindication of surface degradation was detected during the experiment.Subsequently, binding of this set of reference VHHs is also verified tobind monomeric A-beta in multiple ELISA setups (detection described inExample 3), using distinct presentations of peptide formats such ascaptured A-beta(1-40) (C- or N-terminally biotinylated), directlyimmobilized A-beta(1-40) and A-beta(1-42), GST-A-beta(1-42) orA-beta(1-42)-GST (capturing at concentrations of 1 μg/ml). For thereference VHHs 1B, 11G, 8B, 4D, 3A no binding to monomeric A-beta isdetected. In the BAA ELISA only reference VHH 8B show binding toaggregated A-beta at VHH concentrations above 1000 nM. Compared to the2D2 and 2G6 VHHs, the ABII035 and ABII059 VHHs show >50 fold higher EC50values. The results are summarized in Table XIIa.

TABLE XIIa Comparison of binding characteristics of 42D4 and 60A10derived VHHs with binding characteristics of reference anti-A-beta VHHsAggregation BAM BAA inhibition ELISA ELISA (% inhibition EC50 in EC50 inTR-FRET VHH at 26 μM) nM nM IC50 in nM ABII42D4 ND 14.2 0.9 46.8(N-terminal epitope) ABII035 54 (N-terminal epitope) ABII60A10 80 4.90.8 11.1 (central epitope) ABII059 80 (central epitope) 2D2 (central 6915.8 4.7 404*   epitope) 2G6 (N-terminal 29 98.6 7.2 >1000      epitope)1B ND No No No binding binding competition 11G ND No No No bindingbinding competition 8B ND No >1000 No binding nM* competition 4D ND NoNo No binding binding competition 3A ND No No No binding bindingcompetition V31-1 ND No No No binding (C-term epitope) binding bindingFab266 (central 82 1.8 1.8  0.52 epitope) Fab3D6 (N-terminal 82 1.8 0.3 3.4 epitope) ND: not determined; *IC50 calculated from extrapolatedcurves

Example 7 Binding of Anti-Human A-Beta VHHs to Rodent A-Beta (ELISA andBiacore)

Binding of VHHs as obtained above to rodent A-beta is assessed in ELISA.Mouse (Bachem) or human A-beta(1-40) (Anaspec) are coated at 2 μg/mlonto Maxisorp plates and binding of VHHs and, for comparative purposes,Fab fragments, is detected via a mouse anti-myc antibody and ananti-mouse-HRP conjugate (DAKO). EC50 values are determined for the Fabfragment of 3D6, VHH ABII002, and VHH ABII035. The results are listed inTable XIII below (average of minimally two independent experiments).

TABLE XIII EC50 values for binding to human and mouse A-beta humanA-beta(1-40) rodent A-beta(1-40) Clone EC50 (nM) EC50 (nM) ABII002 1.83.3 ABII035 2.5 5.7 Fab3D6 4.2 110

While 3D6 Fab fragments bind mouse A-beta significantly less than humanA-beta (26 fold difference), ABII002 and ABII035 bind equally well toboth (1.8 and 2.2 fold difference, respectively), indicating thatABII002 and its humanized derivative ABII035 recognise an epitope thatis distinct from the epitope recognized by 3D6.

Binding of the VHHs to mouse and human A-beta is further assessed onBiacore with the non-biotinylated peptide coated directly onto the chip.No difference in affinity to human and rodent A-beta peptide is detectedfor the VHHs before and after humanization.

Example 8 Generation and Characterization of Biparatopic Anti-A-Beta VHHConstructs

VHHs ABII42D4 (recognizing the N-terminal region of the A-beta peptide)and VHH ABII60A10 (recognizing the central region) are fused viaflexible glycine-serine linkers (e.g. 9GS: GGGGSGGGG; SEQ ID NO:141) tocreate bivalent VHH constructs. Four biparatopic constructs (comprisingtwo VHH domains with different epitope specificity) differing in linkerlength and orientation are explored in more detail:ABII42D4-25GS-ABII60A10, ABII60A10-25GS-ABII42D4,ABII42D4-35GS-ABII60A10 and ABII60A10-35GS-ABII42D4 and compared torespective VHH dimers (comprising two identical VHH domains, such as thebivalent ABII42D4-9GS-ABII42D4 construct). The biparatopic anddimeric/bivalent VHHs are produced in E. coli TG1 cells and purifiedusing affinity chromatography (IMAC or protein A) and size exclusionchromatography (Superdex75 or Sephacryl S100), resulting in ≧95% purityas assessed via SDS-PAGE.

8.1 TR-FRET Binding Assays:

Binding of the biparatopic VHH constructs to A-beta(1-40) is evaluatedusing the ABII42D4 and ABII60A10 TR-FRET assays (Example 3.2). While thebivalent ABII42D4-9GS-ABII42D4 construct show only a slightly improvedIC50 (3.2 fold improved vs. monovalent ABII42D4), the biparatopic VHHsare surprisingly found to bind significantly stronger to the A-betapeptide than the monovalent building blocks and the 3D6 and m266 Fabfragments (Table XIV) as well as 3D6 and m266 IgG (3D6 and m266full-length monoclonal antibodies). No difference in potency is foundbetween constructs with different linker lengths and orientations.

TABLE XIV IC50 values for biparatopic anti-A-beta VHH constructs andcomparative examples in ABII42D4 and ABII60A10 TR-FRET assays CompetitorIC50 (nM) 42D4 TR-FRET assay ABII42D4-25GS-ABII60A10 0.047ABII42D4-35GS-ABII60A10 0.050 ABII60A10-35GS-ABII42D4 0.055ABII60A10-25GS-ABII42D4 0.043 ABII42D4 51 ABII42D4-9GS-ABII42D4 16ABII60A10 >1000 m266Fab >1000 3D6Fab 17 60A10 TR-FRET assayABII42D4-25GS-ABII60A10 0.066 ABII42D4-35GS-ABII60A10 0.084ABII60A10-35GS-ABII42D4 0.091 ABII60A10-25GS-ABII42D4 0.091ABII42D4 >1000 ABII42D4-9GS-ABII42D4 >1000 ABII60A10 30 m266Fab 2.33D6Fab >1000

8.2 Determination of Binding Mode:

To explore the binding mode of the biparatopic constructs, it isassessed if both VHH building blocks can bind simultaneously to the samepeptide molecule. In a sandwich ELISA, tag-less ABII60A10 or his-taggedABII42D4 are coated onto a Maxisorp plate and incubated with theA-beta(1-40) peptide. The resulting VHH-peptide complex is thenincubated with the c-myc-tagged VHH recognizing the N-terminal orcentral A-beta peptide epitope. Detection via the c-myc is performed asdescribed before (Example 2). Binding is seen for both setups,indicating that the two VHHs recognizing the N-terminal or centralepitope can bind simultaneously to the A-beta peptide.

Determination of the Binding Mode by SPR-Based Assay:

C-terminally biotinylated A-beta(1-40) is immobilized on the sensor chipas described before (Example 2). ABII60A10 is injected at a saturatingconcentration of 500 nM and binding to A-beta(1-40) is observed. After120 seconds, 200 nM of ABII42D4 and 500 nM ABII60A10 are co-injectedresulting in additional binding. A control injection of 500 nM 60A10alone does not result in any additional binding, showing that theadditional binding observed is due to the ABII42D4-A-beta interaction.

Determination of the Binding Mode Using Size Exclusion Chromatography:

Size exclusion chromatography (SEC) separates molecules according todifferences in size as they pass through a gel filtration medium packedin a column. The binding mode of biparatopic VHH constructs to theirtarget can be identified by analysing the protein complex afterincubating a 1:2 molar ratio of the respective VHH construct and thetarget protein. Therefore the mixture will be separated by SEC and thecontents of the resulting peaks is analyzed by protein gelelectrophoresis. An A-beta(1-28)-p38 fusion protein, having a calculatedmolecular weight of 46 kDa, runs as a monomer in the SEC. Thebiparatopic VHH fused to human serum albumin (60A10-27GS-42D4-Alb) has acalculated molecular weight of 95 kDa. In order to identify whether thebiparatopic VHH binds one (=intramolecular binding) or two(=intermolecular binding) A-beta(1-28)-p38 fusion proteins, a 1:2 molarratio of the VHH and the target protein is mixed, incubated over nightat 4° C. and applied to SEC using an ÄKTAexplorer (GE Healthcare, USA)in combination with a preparative size exclusion column (HiLoad 26/60Superdex 200 prep grade, GE Healthcare, USA) in order to separate theprotein complex from the single proteins. The gel filtration producedtwo peaks which are further analyzed by SDS-PAGE using the automatedelectrophoresis station Experion (Bio-Rad, USA) and the appropriate chip(Pro260 Chip, Bio-Rad, USA). Analysis on the Experion reveals that themain peak contains the VHH and the A-beta(1-28)-p38 fusion proteinwhereas the smaller peak contains only the A-beta(1-28)-p38 fusionprotein. The amounts of VHH and A-beta(1-28)-p38 fusion protein in thecomplex are determined using the Experion data analysis software. Themolecular weights of the proteins measured with the Experion are 53 kDafor the A-beta-p38 fusion protein and 101 kDa for the60A10-27GS-42D4-Alb protein. The measured concentrations and thereforecalculated molarity is 0.87 μM and 0.86 μM for the A-beta(1-28)-p38fusion protein and the 60A10-27GS-42D4-Alb protein, respectively.Therefore, the ratio of A-beta(1-28)-p38 to 60A10-27GS-42D4-Alb in theanalysed protein complex is about 1:1 although it is mixed at a molarratio of 1:2. From this result it can be concluded that one biparatopicVHH binds the two epitopes of an A-beta peptide molecule within one andthe same A-beta molecule, and does therefore not (or at least notprimarily) act via cross-linking of A-beta-molecules via the bivalentVHH constructs.

8.3 Crystallization Studies:

For crystallization, ABII035 and ABII059 (160 μM each) are incubatedwith 3-fold molar excess of human A-beta peptide (residues 1-24) for 16hours and the complex is purified by size exclusion chromatography. Thecomplex is concentrated by diafiltration to a concentration of 4 mg/ml.

Crystallization trials are set up as sitting drop vapour diffusionexperiments by mixing 200 nl of protein with 200 nl of reservoirsolution against a reservoir volume of 100 μl. Crystals appear afterseveral days under a variety of different conditions, among these thefollowing is used to determine the structure: 100 mM MMT buffer pH 5,25% PEG1500. Crystals are treated with cryo-protectant (85 mM MMT bufferpH 5, 35% PEG1500) and flash-frozen with liquid nitrogen. Data arecollected at the beamline 6SA of the swiss light source at thePaul-Scherrer Institute in Villigen, Switzerland.

In the crystal structure one molecule of ABII035, ABII059, and a peptidederived from human A-beta peptide (residues 1-24) form a ternarycomplex. This complex dimerizes via an anti-parallel beta-sheet which isformed by two molecules of A-beta peptide. The asymmetric unit of thecrystal is occupied by two of these structures, which totals to fourmolecules of each A-beta peptide, ABII035 and ABII059. Residues 1 to 9of A-beta adopt an alpha-helical conformation, residues 10 to 20 form abeta-strand. Residues 1 to 14 of A-beta are in proximity to ABII035 andamong these Asp1, Ala2, Glu3, Phe4, Asp7, and His14 directly contactABII035. Residues 15 to 24 of A-beta are in proximity to ABII059 andamong these Gln15, Lys16, Val18, Phe19, Phe20, Glu22, and Asp23 directlycontact ABII059.

In addition to the above, it becomes clear from the crystal structurethat ABII035 and ABII059 can bind to one and the same molecule of A-betasimultaneously, confirming the results as obtained in Example 8.2 above.

Furthermore, the mode of interaction of ABII035 with the A-beta derivedpeptide as evidenced by the above crystal structure data is consistentwith the observation as described in Example 7 above: ABII035 shows agood species cross-reactivity with regard to rodent A-beta peptides. Theonly sequence differences between human and rodent A-beta are R5G, Y10Fand H13R. According to the above data, none of these residues formcontacts to the VHH. Therefore it is assumed that rodent A-beta can bindto ABII035 in the same conformation as human A-beta, making itparticularly useful as a tool reagent (research tool) for assaysinvolving rodent animal models (producing rodent A-beta peptide), suchas mice or rats.

Example 9 Generation and Characterization of Half-Life ExtendedHumanized Biparatopic Anti-A-Beta VHH Constructs

9.1 Generation of constructs ABII314 to ABII323:

For in vivo validation studies with humanized biparatopic VHHconstructs, different half life extension (HLE) strategies areexplored: 1) genetic fusion to an albumin binding VHH (as described e.g.in WO2004/041865), 2) PEGylation of a Cys residue located in the linkerbetween VHHs or at the C-terminus of the VHHs (WO2008/142164) and 3)genetic fusion to human or mouse serum albumin. The different HLEstrategies are explored with biparatopic VHH constructs comprising theABII035 and the ABII059 VHH domains/building blocks. Constructs ABII314to ABII323 are generated via gene assembly using appropriate sets ofoverlapping oligonucleotides. Sequence IDs and amino acid sequences ofABII314 to ABII323 are listed in Table XV. In constructs ABII314,ABII315, ABII322 and ABII323, building block ABII059 is fused to ABII035via a Gly-Ser linker of different length (as indicated in Table XV),containing a cysteine residue for conjugation with PEG (at the positionas indicated in Table XV). Constructs ABII316 to ABII321 consist ofgenetically linked VHHs ABII035, ABII059 and ALB8 (humanized anti-humanalbumin VHH with mouse albumin cross reactivity), in differentorientations and separated by either 9- or 35-GS linkers. The constructsshown in Table XV below may optionally additionally include ahexa-histidine tag and/or other tags for e.g. facilitating purificationof the resulting polypeptides.

TABLE XV  Biparatopic VHH constructs ABII314 to ABII323 SEQ ID CloneSequence information Description NO: ABII314EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-35GSC 142WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS with C atRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR positionRSYSSWGQGTLVTVSSGGGGCGGGGSGGGGSGGG 5-035GSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGS LRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS ABII315EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-9GSC 143WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS with C atRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR positionRSYSSWGQGTLVTVSSGGGGCGGGSEVQLLESGGG 5-035LVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQG TLVTVSS ABII316EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-9GS- 34WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS Alb8-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINV 035RRSYSSWGQGTLVTVSSGGGGSGGGSEVQLVESGG GLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGG GGSGGGSEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTANNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHR FVVGGNRVEDWRYWGQGTLVTVSS ABII317EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-35GS- 35WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS 035-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINV Alb8RRSYSSWGQGTLVTVSSGGGGSGGGGSGGGGSGG GGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRP EDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAAS GFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSS ABII318EVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMG 059-9GS- 36WFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTIS 035-9GS-RDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINV Alb8RRSYSSWGQGTLVTVSSGGGGSGGGSEVQLLESGG GLVQPGGSLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTANNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWG QGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVTVSS ABII319EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-35GS- 37WFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTIS 059-9GS-RDNSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNR Alb8VEDWRYWGQGTLVTVSSGGGGSGGGGSGGGGSG GGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVS SGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI GGSLSRSSQGTLVTVSS ABII320EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGW 035-9GS- 38FRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRD Alb8-9GS-NSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVE 059DWRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGG LVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNY NMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAI NVRRSYSSWGQGTLVTVSS ABII321VQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMGWF 035-9GS- 39RQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRD Alb8-9GS-NSKNTAYLQMNSLRPEDTAVYYCAAHRFVVGGNRVE 059DWRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGG (first ELVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLE deleted)WVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNY NMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAI NVRRSYSSWGQGTLVTVSS ABII322VQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGW 059-27GSC 40FRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRD with C atNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRS positionYSSWGQGTLVTVSSGGGSGGGGSGGGGC*GGGGS 14-035GGGGSGGGEVQLLESGGGLVQPGGSLRLSCVHSGP TFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAAHR FVVGGNRVEDWRYWGQGTLVTVSS ABII323VQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGW 059-35GSC 41FRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISR with C atDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVR positionRSYSSWGQGTLVTVSSGGGGSGGGGSGGGGC*GG 15-035GGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGG SLRLSCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQMNSLRP EDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSS

9.2 Production, Purification and PEGylation of Humanized Half-LifeExtended Biparatopic VHH Constructs:

His₆-tagged biparatopic VHH constructs ABII314, ABII315, ABII322 andABII323 are purified via affinity chromatography (IMAC or protein A),cation exchange chromatography (SP Sepharose, Source 30S or POROS 50HS)and size exclusion chromatography (Superdex75 or Sephacryl S100). Atprotein purity levels of minimally 90% (determined via SDS-PAGE andsubsequent Coomassie Brilliant Blue staining), PEGylation is performed.

Intermolecular S—S bonds of VHHs are first reduced by addingDL-Dithiothreitol solution (DTT) to a final concentration of 10 mMfollowed by 2 to 3 h incubation at room temperature or overnight at 4°C. The reducing agent is subsequently removed via size exclusionchromatography (Superdex 75/200) in D-PBS running buffer, collecting themain peak. Immediately after removing the reducing agent, thebiparatopic polypeptide construct is incubated with a 5fold molar excessof PEG for 1 h at room temperature or overnight at 4° C. The PEGylatedpolypeptide is separated from free PEG and non-PEGylated polypeptide viacation exchange chromatography (MacroCap SP). A final polishing step andbuffer change to D-PBS is performed via preparative size exclusionchromatography on Superdex200 or Sephacryl S200. The quality of thepurified PEGylated polypeptide is verified via SDS-PAGE followed by CBB-and PEG-staining as described before by Natarajan et al (1995) inBioconjugate chemistry 16:113-121.

9.3 Characterization of Half-Life Extended Humanized BiparatopicAnti-A-Beta VHH Constructs by TR-FRET:

In order to compare the binding properties of the half-life extendedbiparatopic VHH constructs described above to those of monoclonalantibodies (IgG) 3D6 and m266, ABII315 to ABII320, ABII322 and ABII323are tested in ABII002 and ABII050 competition TR-FRET assays (seeExamples 8.1 and 3.2). Average IC50s derived from at least twoindependent experiments are summarized in Table XVI.

TABLE XVI IC50 values for half life extended anti-A-beta biparatopicVHHs in absence/presence of albumin. IC50 (nM) IC50 (nM) VHH ABII002assay ABII050 assay Monovalent VHHs and Fabs ABII002 2.9 NC ABII035 3.5NC ABII050 NC 6.3 ABII059 NC 12.8 Fab3D6 3.4 NC Fab266 NC 0.52Multivalent VHHs and IgGs ABII315-PEG20 0.13 0.28 ABII315-PEG40 Nottested 0.31 ABII322-PEG40 0.14 0.17 ABII323-PEG40 0.18 0.20 ABII316 0.190.31 ABII316 + 10 μM HSA 0.05 0.06 ABII317 0.29 0.49 ABII318 0.13 0.28ABII319 0.26 0.33 ABII320 0.25 0.21 ABII059-27GS-ABII035-MSA 0.19 0.18ABII059-27GS-ABII035- 0.10 0.12 MSA_D3 IgG 3D6 1.9 NA IgG m266 NA 0.53NC: no competition detected MSA: mouse serum albumin MSA_D3: domain IIIof mouse serum albumin

In the ABII002 TR-FRET competition assay, all half-life extended VHHconstructs tested show a highly improved potency between 6.5fold and14.6fold (for ABII315-PEG20) compared to 3D6 IgG (IC50 of 1.9 nM). Whencompared to IgG m266, all VHH constructs show significantly better IC50values in the ABII050 TR-FRET competition assay, with VHH ABII320 havingthe lowest IC50 of 0.21 nM, 2.5fold improved over IgG266.

Compared to the potency of monovalent VHH ABII035, the tailored VHHconstruct with the highest potency (ABII315-PEG20) show a 27foldimproved IC50 in the ABII002 TR-FRET competition assay. VHH constructABII320 show a 61-fold improved potency over monovalent ABII059 in theABII050 TR-FRET competition assay.

The increased potency of the biparatopic constructs over the monomericbuilding blocks ABII035 and ABII059 confirms the results obtained withthe non-humanized, non-matured constructs (Example 8.1) and supports thehypothesis of intramolecular binding of one biparatopic VHH construct toone A-beta peptide molecule (cf. Example 8.2 above). Furthermore, theseexperiments demonstrate that the half-life extension techniques appliedto the VHH constructs as described above do not interfere with bindingof the constructs to the A-beta peptide.

To assess whether the binding of ALB8-containing VHH constructs to theA-beta peptide is affected by HSA (as present in the bloodstream),biparatopic constructs equivalent to ABII316-ABII320 but containing thenon-humanized VHH building blocks are tested in both competition TR-FRETassays in presence and absence of μM amounts of albumin. In both assayformats, pre-incubation with 6.5-10 μM human, dog or bovine albumin (thelatter two albumin variants showing no detectable interaction with ALB8)do not affect potency in the TR-FRET assays.

To test whether ALB8 can still bind to albumin in the context of thebiparatopic VHH constructs, a kinetic analysis of the VHH constructs forbinding to a chip coated with HSA is performed (Biacore). Comparableaffinities were determined for monovalent ALB8 and the ALB8 fusionconstruct tested, indicating that HSA-binding of the ALB8 building blockis not affected.

9.4 Inhibition of A-Beta Fibril Formation:

In a further experiment, a dilution series of the biparatopic VHHconstruct ABII320 is evaluated for its capacity to inhibit formation ofA-beta fibrils in vitro as described under Example 3.5 and is comparedto the activity of its monovalent building blocks ABII035 and ABII059and control antibodies. The maximum inhibition at the highestconcentration of the VHH construct tested (26 μM) is calculated as anaverage of two independent experiments. While a consistent backgroundinhibition of approximately 17% is detected when using non-related(non-A-beta specific) VHHs as a control, monovalent 3D6 and m266 Fabfragments show a reproducible dose-dependent inhibition with maximalinhibition of 82%. (Intact) IgGs 3D6 and m266 result in 75% and 83%inhibition, respectively. Monovalent VHHs ABII035 and ABII059 show amaximum inhibition of 54% and 80%, respectively, while the half-lifeextended biparatopic VHH construct ABII320 shows a maximum inhibitioncapacity of 84% at 26 μM.

9.5 Binding to Soluble APP-Alpha:

Soluble APP-alpha (sAPPalpha) is released from its cell bound precursorprotein APP by alpha-secretase activity. The amino acid sequence ofsAPPalpha contains the first sixteen amino acid residues of the A-betapeptide, but does not contain the correct 3D6 epitope, as a freeN-terminal amino acid residue in the A-beta peptide is essential forfull 3D6 interaction. Epitope mapping indicates that N-terminalregion-specific VHH ABII001 and its derivatives interact with a distinctepitope compared to monoclonal antibody 3D6. VHH-binding to sAPPalpha incomparison to A-beta is tested using a TR-FRET-based assay setup.Biotinylated sAPPalpha (Sigma) at a final concentration of 0.82 nM ismixed with the same concentration of streptavidin-Europium labeled beadsand 4.4 nM of AlexaFluor647-labeled ABII320. The following non-labeledcompounds are tested for competition: ABII320, sAPPalpha, IgG m266, IgG3D6, ABII035 and A-beta(1-40). Two independent experiments are performedapplying a dilution series of the competitor compounds. Average IC50values are 0.72 nM, 25.6 nM, 0.23 nM, and 14 nM for non-labeled ABII320,ABII035, A-beta(1-40) and sAPPalpha, respectively. As expected, nocompetition is observed for IgG m266 and IgG 3D6. In this assay set-up,ABII320 interacts 63-fold better with A-beta(1-40) than with sAPPalpha.In a parallel set-up using biotinylated A-beta(1-40) instead ofbiotinylated sAPPalpha, ABII320 interacts at least 20000-fold betterwith A-beta(1-40) than with sAPPalpha (IC50 estimated based onextrapolated data at higher concentrations), indicating that ABII320prefers binding to A-beta(1-40) over sAPPalpha.

9.6 Epitope Mapping for Biparatopic VHH Constructs:

A-beta(1-42) derived peptides displayed as peptide microarrays(PepStar™) are used to determine the epitopes of VHHs. PepStar™ peptidemicroarrays (JPT Peptide Technologies, Germany) are customized peptidemicroarray sets for rapid screening of antibody/VHH peptide interactiondisplayed on glass slides. The peptides of the microarray represent a12/11 scan derived from the primary structure of A-beta(1-42)immobilized via their N-terminus. In addition, truncated peptides of theN-terminus representing the first 16 to the first 7 amino acids ofA-beta(1-42) are immobilized via their C-terminus. Upon incubation withthe VHH, the binding event can be detected by reading the fluorescenceintensity of labeled secondary antibody directed against the VHH.

After blocking the microarray with blocking buffer (PBS, 1% BSA, 0.1%Tween20) over night at 4° C., it is incubated for 2 h at 4° C. with 200μl of the biparatopic VHH construct ABII320 in wash buffer (PBS, 5 mMDTT, 0.05% Triton X-100, 5% Glycerol, 1% BSA) at a concentration of 5μg/ml. In order to remove excess VHH, the microarray is incubated threetimes for two minutes on ice with wash buffer. Thereafter, themicroarray is incubated for one hour at 4° C. with an anti-VHH AlexaFluor 647 (Boehringer Ingelheim Pharma GmbH & Co KG, Germany) in washbuffer at a concentration of 0.5 μg/ml. The anti-VHH is derived from agoat serum after immunization with the VHH dimer G6-G6. Unboundsecondary antibody is removed by incubating the microarray three timesfor two minutes on ice with wash buffer. Finally the slide is dried in acentrifuge at 800×g for three minutes and scanned with the ProScanArrayfrom Perkin Elmer using a 633 nm laser. The data are analyzed with theScanArray Express® software from Perkin Elmer.

As a result, peptide spots having a diameter of 420 μm are obtainedwhich are analyzed for fluorescence intensity, from which interaction ofthe VHH with the peptides can be assessed in a semi-quantitative manner.Thus, incubation of the biparatopic VHH construct ABII320 with thepeptide microarrays reveals that ABII320 interacts with residues 1-11and 15-24 of the A-beta peptide. The alanine scan indicated thatresidues Asp1, Glu3, Phe19, Phe20 and Asp23 of the A-beta peptide areessential for the binding of the biparatopic VHH, which is consistentwith the data obtained from crystal structure analyses. The same resultsare obtained using two other biparatopic VHH constructs which includethe ABII035 and ABII059 VHH domains.

9.7 Determination of Binding Constants Using Kinetic Exclusion Assay(KinExA):

Affinities of three biparatopic VHH constructs for the A-beta peptideare determined using KinExA (Kinetic Exclusion Assay). KinExA is atechnology with the ability to measure unmodified molecules in solutionphase (Darling, R. J. and Brault, P.-A.: Kinetic exclusion assaytechnology: Characterization of Molecular Interactions; Assay and DrugDevelopment Technologies 2(6):647 (2004)). The KinExA method measuresthe concentration of uncomplexed VHH molecules in a mixture of VHH,antigen, and VHH-antigen complex. The concentration of uncomplexed VHHis measured by exposing the solution phase mixture to solid phaseimmobilized antigen for a very brief period of time. The “contact time”between the solution phase mixture and the solid phase immobilizedantigen is kept short enough that dissociation of VHH-antigen complex isinsignificant. When the possibility of significant dissociation ofVHH-antigen complex is kinetically excluded, only uncomplexed (“free”)VHH can bind to the solid phase. The amount of free VHH that binds tothe solid phase (measured by fluorescence emission from a secondarylabel) is directly proportional to the concentration of free VHH in thesolution phase sample.

A-beta(1-28) peptide (Anaspec, USA) is used as antigen for theincubation with biparatopic VHHs. Fusion protein“His.Xa.Abeta1-28.GS.p38” is used to prepare the solid phase for bindingthe uncomplexed VHHs. The structure of the fusion protein is as follows:His-tag, factor Xa cleavage site, A-beta(1-28) peptide, GS-linker,murine p38 alpha and has the following sequence:

(SEQ ID NO: 144) MHHHHHHIEGRDAEFRHDSGYEVHHQKLVFFAEDVGSNKGGSGGSQERPTFYRQELNKTIWEVPERYQNLSPVGSGAYGSVCAAFDTKTGHRVAVKKLSRPFQSIIHAKRTYRELRLLKHMKHENVIGLLDVFTPARSLEEFNDVYLVTHLMGADLNNIVKCQKLTDDHVQFLIYQILRGLKYIHSADIIHRDLKPSNLAVNEDCELKILDFGLARHTDDEMTGYVATRWYRAPEIMLNWMHYNQTVDIWSVGCIMAELLTGRTLFPGTDHIDQLKLILRLVGTPGAELLKKISSESARNYIQSLAQMPKMNFANVFIGANPLAVDLLEKMLVLDSDKRITAAQALAHAYFAQYHDPDDEPVADPYDQSFESRDLLIDEWKSLTYDEVISFVPPPLDQEEMES.

The fusion protein is expressed in E. coli, purified using Ni-NTA(Qiagen, Germany) and alkylated with Iodoacetamide (Sigma, USA).Polymethylmethacrylate beads (Sapidyne Instruments Inc., USA) are coatedwith the fusion protein in PBS with 0.02% NaN₃, blocked with 1% BSA inthe same buffer and used for all experiments as solid phase. The KinExAexperiments are conducted with a KinExA 3000 and the KinExA Pro software(Sapidyne Instruments Inc., USA) at room temperature (ca. 21° C.). PBSwith 0.02% NaN₃ is used as running buffer. For all experiments, antigenis serially diluted into PBS with 0.1% BSA and 0.02% NaN₃ having aconstant VHH concentration. The mixtures of antigen and VHH areincubated for 24 h at room temperature prior to measurement. The flowrate of the samples and the labeling antibody for all experiments is0.25 ml/min.

The secondary fluorescence labeled antibody for the measurement of theABII320 VHH construct (first VHH construct) is the Alexa 647-conjugatedgoat anti-G6G6 (anti-VHH) described under 9.6 above. This antibody isused at a volume of 500 μl and a concentration of 0.5 μg/ml in PBS with0.1% BSA and 0.02% NaN₃. The ABII320 is additionally measured asdescribed in the presence of recombinant human albumin (Albcult™,novozymes, USA). Binding of the PEGylated ABII322 VHH construct (secondVHH construct) to the solid phase is determined using 500 μl of a rabbitanti-PEG (Epitomics Inc., USA) at a concentration of 0.1 μg/ml in PBSwith 0.1% BSA and 0.02% NaN₃ on line 13 of the instrument in combinationwith 250 μl of an Alexa 647-conjugated goat anti-rabbit (MolecularProbes Inc., USA) at a concentration of 0.25 μg/ml in the same buffer.Binding of the HSA-biparatopic VHH construct fusion protein shown inTable IV above (SEQ ID NO:32; third VHH construct) to the solid phase isdetermined using 500 μl of a goat anti-HSA (Bethyl Laboratories Inc.,USA) at a concentration of 0.5 μg/ml in PBS with 0.1% BSA and 0.02% NaN₃on line 13 of the instrument in combination with 500 μl of an Alexa647-conjugated rabbit anti-goat (Molecular Probes Inc., USA). at aconcentration of 0.5 μg/ml in the same buffer.

The equilibrium titration data are fit to a 1:1 binding model usingKinExA Pro software Version 1.0.3 (Sapidyne Instruments Inc., USA). Thefirst VHH construct (biparatopic VHH ABII320) is measured atconcentrations of 3, 5 and 10 pM. The sample volume is 5 ml each. Themeasured K_(D) is 1.1±0.3 pM and 0.4±0.1 pM in the presence of 1%recombinant human albumin. The second VHH construct (biparatopicPEGylated VHH ABII322) is measured at concentrations of 3, 4, 5 and 6pM. The sample volume is 11 ml each. The measured K_(D) is 0.6±0.2 pM.The third VHH construct (HSA-VHH fusion protein) is measured atconcentrations of 2, 3 and 4 pM. The sample volume is 5 ml each. Themeasured K_(D) is 0.5±0.3 pM.

9.8 Binding to Pyroglutamyl-Abeta:

Pyroglutamyl (pGlu)-A-beta is a major component of neuritic plaques inAlzheimer's disease. It is formed by cyclization of the N-terminalglutamate at position 3 catalyzed by glutaminyl cyclase (QC) resultingin a very amyloidogenic variant of A-beta. The amino acid sequence ofpGlu-A-beta contains the amino acid residues 3-42 of the A-beta peptide.The first two N-terminal amino acids which are essential for fullinteraction with antibody 3D6 are missing. Affinities for thepGlu-A-beta-ABII320/ABII322/m266/3D6 interaction are determined viasurface plasmon resonance (Biacore) using C-terminally biotinylatedpGlu-A-beta(1-28) captured on a streptavidin sensor chip as describedbefore (Example 2). Purified polypeptides ABII320 or ABII322 orantibodies m266 or 3D6 are injected at 9 different concentrations(between 0.195 and 50 nM for ABII320, 0.39-100 nM for ABII322 and0.782-200 nM for 3D6 and m266) for 3 min and allowed to dissociate for10 min. Association and dissociation curves can be fitted using a 1:1interaction model and an accurate K_(D) value can be determined. Theaffinities are found to be 2 nM for ABII320, 1 nM for ABII322, 224 pMfor the antibody m266 and no binding could be observed for the antibody3D6.

Example 10 Binding of Anti-A-Beta VHHs to Amyloid Plaques

VHHs are profiled in a tissue amyloid plaque immunoreactivity (TAPIR)assay according to their capability to bind to amyloid plaques in Tg2576mouse brain slices. The TAPIR assay allows a qualitative (dense coreand/or diffuse amyloid plaques) as well as quantitative assessment(minimum effective dosis MED) of the VHHs. For the assay, cryostat-cutcoronal sections of plaque-positive mice are prepared, mounted onto76×26 mm Superfrost slides (Roth) and stored at −20° C. Cryosections arethawed for 20 minutes, fixed in 3% paraformaldehyde (PFA) for 10 minuteson ice. Sections are then incubated in 0.3% hydrogen peroxide for 30minutes, blocked with PBS containing 2.5% bovine serum albumin (BSA) and2% mouse serum for 1.5 hours and incubated with a myc-tagged VHH or Fabat different concentrations (3.0 mg/ml to 4.11 ng/ml), dissolved in PBScontaining 2.5% BSA. The slices are then incubated for 1.5 hours with 10mg/ml biotinylated mouse anti c-myc monoclonal antibody (9E10)(Sigma-Aldrich), dissolved in PBS containing 2.5% BSA (except for slicesincubated with biotinylated ABII320). The sections are incubated for 1.5hours with Vectastain ABC ELITE Kit solution (Vector Laboratories)prepared according to manufacturer's instructions, and visualised usingVector VIP SK 4600 solution (Vector Laboratories), again preparedaccording to manufacturer's instruction. The sections are progressivelydehydrated by 5 minute incubations in 60-, 80- and 100% ethanol and 100%isopropanol and cleared for 5 minutes in xylene. The sections are thenmounted with Entellan new mounting medium (Merck) and covered with 24×50mm coverslips (Menzel-Glaeser). The results are summarized in thefollowing table.

In addition to ABII320 and Fab fragments of monoclonal antibody m266described above, several other (myc-tagged) biparatopic VHH constructswere analysed in the above TAPIR assay. The structure of VHH constructsABII300 to ABII305 is shown in Table XVII below and, in all cases,includes VHH domains ABII002 (SEQ ID NO:62) and ABII050 (SEQ ID NO:100),in different orders and combined with either an albumin binding VHHdomain (ABII300 to ABII304) or a PEG40 moiety (ABII305).

From the results, it can be seen that biparatopic VHHs ABII300, ABII301,ABII302, ABII303 and ABII304 as well as ABII305-PEG40 reveal the sameaffinity to bona fide tissue amyloid plaques (dense core and diffusetype of plaques) in the TAPIR assay. A particular order of the linkersor conjugation to PEG40 does not appear to have a significant effect onplaque affinity.

TABLE XVII Structure of biparatopic VHH constructs ABII300 to 305 andresults obtained in TAPIR assay VHH Domain Structure Epitope MED [ug/ml]m266 central no binding (Fab) ABII300 ABII050-GS9-Alb8-GS9-ABII002N-term and 0.11 > x > 0.04 central ABII301 ABII050-GS35-ABII002-GS9-Alb8N-term and 0.11 > x > 0.04 central ABII302 ABII050-GS9-ABII002-GS9-Alb8N-term and 0.11 > x > 0.04 central ABII303 ABII002-GS35-ABII050-GS9-Alb8N-term and 0.11 > x > 0.04 central ABII304 ABII002-GS9-Alb8-GS9-ABII050N-term and 0.11 > x > 0.04 central ABII320 ABII035-GS9-Alb8-GS9-ABII059N-term and 0.11 > x > 0.04 central ABII305- ABII050-GSC35(PEG at Cys atN-term and 0.11 > x > 0.04 PEG40 position 5)-ABII002 central

Example 11 Pharmacodynamic of Half-Life Extended Biparatopic Anti-A-BetaVHH Constructs

To determine the pharmacokinetic and pharmacodynamic properties of VHHs,samples are injected either i.v. or i.p. into APP transgenic mice.Plasma samples are taken by bleeding the V. saphena. A predose samplewas taken before the VHHs, IgGs or vehicle application and after 4 and24 hours.

As the binding of the VHHs and the IgGs interferes with the detection inthe ELISA, the A-beta:VHH or A-beta:IgG complexes are denatured prior tothe assay in order to detect the total amount of plasma A-beta(1-40). Inbrief, samples are denatured with 6M GuHCl (Sigma Aldrich) and purifiedover a solid-phase extraction column. 60 mg Oasis HLB 96-well plates(Waters) are set into extraction plate manifolds (Waters) connected tohouse vacuum. Columns are activated with 1 mL methanol (MeOH), followedby 1 mL H₂O. GuHCl-extracted samples are loaded and washed sequentiallywith 1 mL volumes of 5 and 30% MeOH and A-beta is then eluted with 2%NH₄OH in 90% MeOH. Eluted samples are collected and vacuum-centrifuged(Eppendorf Vacufuge) at 1400 rpm, 60° C. for 90 min. Once samples aredried completely, they are reconstituted in blocking buffer and storedat −20° C. until analysis. Total plasma levels of A-beta(1-40) aredetermined by sandwich ELISA (4G8/anti-A-beta(1-40), mesoscalediscoveries) according to the manufacturer's instruction.

Predose total A-beta(1-40) levels for APP transgenic mice areapproximately 1.25 nM in plasma. In these mice injected with 15 nmol/kgof biparatopic VHHs or IgG (same dose, normalized to binding sites) thelevel of total A-beta in plasma peaked around 4 hours after i.v.application. Data are expressed as ‘fold increase over predose’ in whichthe A-beta(1-40) plasma concentrations determined at 4 and 24 hrs afterinjection are divided by the predose levels. Results are shown in TableXVIII.

TABLE XVIII Fold increase and AUD of plasma A-beta(1-40) after i.v.injection of anti-A-beta VHHs, compared to anti-A-beta antibodies 3D6and m266 plasma A-beta(1-40) fold increase over predose APP transgenicmice dose exp. (nmol/kg) 4 hrs 24 hrs AUD ABII300 14/16 15 i.v. 37.7821.00 632.5 ABII301 14/16 15 i.v. 26 13.34 402.9 ABII305 16 15 i.v.33.86 15.81 877.5 ABII306 21 15 i.v. 45.05 32.83 862.5 ABII316 24-26 15i.v. 21.88 9.26 649 ABII317 24-26 15 i.v. 35.91 18.95 1615 ABII318 24-2615 i.v. 13.45 5.81 807.9 ABII319 24-26 15 i.v. 36.53 21.53 1313 ABII32024-26 15 i.v. 42.36 17.57 969.9 ABII315- 24-26 15 i.v. 28.78 12.14 761PEG40 m266 IgG1 20  7.5 i.v. *  25.79 17.9 528.9 3D6 IgG2b 20  7.5 i.v.*  29.47 25.88 673.2 * 15 nmol/kg, when calculated for binding sites (2per IgG molecule)

Thus, compared to the IgG molecules known in the art, biparatopic VHHconstructs have the potential to show up to 50% higher peak levels fortotal plasma A-beta (4 hrs) and >2 fold increased AUD values, indicatingsuperiority of these VHH constructs over the IgGs in capturing A-beta.

To confirm that the level of free/unbound plasma A-beta decreasessubsequent to administration of the VHH construct, a competitive ELISAis developed. Briefly, A-beta(1-40) is captured with anA-beta(1-40)-specific antibody coupled to an ECL-ELISA plate (mesoscalediscoveries). The VHH or IgG molecules that are used to treat theanimals in passive immunotherapy are tagged using the MSD Sulfo-TagNHS-Ester (mesoscale discoveries) according to the manufacture's manualand used as detection tool in this sandwich ELISA format. A-beta(1-40)that is bound to the VHH or to IgG can not be detected in these settingswhereas free/unbound A-beta(1-40) is detectable. To compare data fromdifferent assays, all values are normalized to the data from a vehicletreated group. APP transgenic mice are treated with VHHs or IgGs asindicated and plasma is collected and analyzed for free/unboundA-beta(1-40). As soon as 2 hours after i.p. injection of 132 nmol/kganti-A-beta VHHs (ABII320 and ABII322, respectively), the levels offree/unbound A-beta(1-40) decreased strongly and highly significant frombaseline levels (appr. 2 nM) down to below the detection limit at 2 pM.Equivalent dose of 3D6-IgG (10 mg/kg, 132 nmol/kg binding sites=66nmol/kg IgG) is able to reduce unbound A-beta(1-40) in plasma only downto a level of 52 pM (FIG. 1). In this comparison the biparatopic VHHconstructs ABII320 and ABII322 show, at the same dose, an unexpectedlymuch stronger decrease in free/unbound A-beta in plasma compared to 3D6IgG, again indicating superiority in A-beta capture over IgG (3D6). Thismakes such VHH constructs particularly useful in terms of therapeuticefficacy of VHHs. Specifically, a more efficient depletion of A-beta inplasma is expected to completely prevent the influx of A-beta fromplasma into the brain, thereby generating a steeper concentrationgradient between the brain and plasma A-beta pools, thereby acceleratingefflux of A-beta from the brain into the plasma.

Example 12 BBB Crossing VHHs

In order to further improve the blood brain barrier (BBB) crossingcharacteristics of the VHH constructs, bispecific VHHs are constructedcomprising the biparatopic VHHs and the BBB crossing VHHs FC44 and FC5(as described in detail in WO2002/057445). These VHHs improve the BBBcrossing by binding to a target protein expressed on the BBB, afterwhich the VHHs are transcytosed through the endothelial cells andreleased in the brain parenchyme. As such, VHHs genetically fused to theFC44 and FC5 VHHs are expected to undergo an active transport into thebrain, resulting in higher brain levels and improved therapeutic effect.Constructs ABII400-ABII407, comprising the FC44 or FC5 VHHs, aregenerated via gene assembly using appropriate sets of overlappingoligonucleotides. SEQ ID NOs and amino acid sequences are listed inTable XIX below. In summary, VHHs are generated that contain the FC5 orFC44 VHH either between the ABII035 and ABII059 VHH building, or betweenthe HSA binding VHH and ABII035 or ABII059 building blocks, or at theamino or carboxy-terminal end of the molecule (see Table XIX). VHHs areexpressed in E. coli or in Pichia pastoris and purified as described inExample 9.2. Purified VHHs are assessed in an in vitro BBB crossingassay as described (WO2002/057445 and further references therein). Inaddition, radiolabeled or non-radiolabeled VHHs comprising the FC5 andFC44 building blocks are administered in vivo and the brain levels aredetermined using either liquid scintillation counting or ELISA.

TABLE XIX  Biparatopic anti-A-beta VHH constructs, additionallycomprising FC5 and FC44 moieties VHH construct  domain SEQ ID structureID NO: ABII400 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-9GS- 145WFRQAPGKGREFVAAVTWNSGRINY FC5-Alb8- ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYC9GS-059 AAHRFVVGGNRVEDWRYWGQGTLV TVSSGGGGSGGGSEVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREF VSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPL RVDYWGKGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNY NMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTA VYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS ABII401EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-9GS- 146WFRQAPGKGREFVAAVTWNSGRINY Alb8-9GS- ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCFC44-9GS- AAHRFVVGGNRVEDWRYWGQGTLV 059TVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLS CAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMN SLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLQASGGGLVQAGGS LRLSCSASVRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFVKGRFSISRDNAKNMV YLQMNSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVTVSSGGGGSGGGSEVQ LLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADS VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS ABII402EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-9GS- 147WFRQAPGKGREFVAAVTWNSGRINY Alb8-9GS- ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYC059-9GS- AAHRFVVGGNRVEDWRYWGQGTLV FC5 TVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEW VSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGK GREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGT AINVRRSYSSWGQGTLVTVSSGGGGSGGGSEVQLQASGGGLVQAGGSLRLSCAASGFKIT HYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPED TADYYCAAGSTSTATPLRVDYWGKGTQVTVSS ABII403EVQLQASGGGLVQAGGSLRLSCSASVRTFSIYAMG FC44-9GS- 148WFRQAPGKEREFVAGINRSGDVTKY 035-9GS- ADFVKGRFSISRDNAKNMVYLQMNSLKPEDTALYYCAlb8-9GS- AATANAYDTVGALTSGYNFWGQGTQ 059VTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRL SCVHSGPTFRTDTMGWFRQAPGKGREFVAAVTWNSGRINYADSVKGRFTISRDNSKNTAYLQ MNSLRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSSGGGGSGGGSEVQLVES GGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQL LESGGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADS VKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSS ABII404EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-35GS- 149WFRQAPGKGREFVAAVTWNSGRINY 059-9GS- ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCAlb8-9GS- AAHRFVVGGNRVEDWRYWGQGTLV FC44 TVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSSGGGGSGGGSEVQLVESG GGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQL QASGGGLVQAGGSLRLSCSASVRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFV KGRFSISRDNAKNMVYLQMNSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVTVS S ABII405 EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG035-9GS- 150 WFRQAPGKGREFVAAVTWNSGRINY FC44-9GS-ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYC 059-9GS- AAHRFVVGGNRVEDWRYWGQGTLVAlb8 TVSSGGGGSGGGSEVQLQASGGGLVQAGGSLRLS CSASVRTFSIYAMGWFRQAPGKEREFVAGINRSGDVTKYADFVKGRFSISRDNAKNMVYLQM NSLKPEDTALYYCAATWAYDTVGALTSGYNFWGQGTQVTVSSGGGGSGGGSEVQLLES GGGLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSSGGGG SGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSG SDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS ABII406EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMG FC5-9GS- 151WFRQAPGKEREFVSRITWGGDNTFY 035-9GS- SNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYC059-9GS- AAGSTSTATPLRVDYWGKGTQVTV Alb8SSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCV HSGPTFRTDTMGWFRQAPGKGREFVAAVTANNSGRINYADSVKGRFTISRDNSKNTAYLQMNS LRPEDTAVYYCAAHRFVVGGNRVEDWRYWGQGTLVTVSSGGGGSGGGSEVQLLESGG GLVQPGGSLRLSCAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISR DNSKNTVYLQMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSSGGGGSGG GSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDT LYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS ABII407EVQLLESGGGLVQPGGSLRLSCVHSGPTFRTDTMG 035-35GS- 152WFRQAPGKGREFVAAVTWNSGRINY 059-9GS- ADSVKGRFTISRDNSKNTAYLQMNSLRPEDTAVYYCFC5-9GS- AAHRFVVGGNRVEDWRYWGQGTLV Alb8 TVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLS CAASGRTFNNYNMGWFRQAPGKGREFVAAVSRSGVSTYYADSVKGRFTISRDNSKNTVYL QMNSLRPEDTAVYYCAAAYRGTAINVRRSYSSWGQGTLVTVSSGGGGSGGGSEVQLQASG GGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITANGGDNTFYSNSVKGRE TISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSSGGGGSG GGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSD TLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS

Example 13 Industrial Manufacturing Process for PEGylated BiparatopicPolypeptides of the Invention

13.1 Fermentation:

Any of the polypeptides ABII305, ABII306, ABII314, ABII315, ABII322, andABII323 can be expressed in the cytoplasm of different E. coli strainslike W3110, TG1, BL21, BL21(DE3), HMS174, HMS174(DE3), MM294 undercontrol of an inducible promoter. This promoter can be chosen fromlacUV5, tac, T7, trp, T5, araB. The cultivation media are preferablyfully defined according to Wilms et al., 2001 (Wilms, B., Hauck, A.,Reuss, M., Syldatk, C., Mattes, R., Siemann, M., and Altenbuchner, J.:High-Cell-Density Fermentation for Production of L-N-Carbamoylase Usingan Expression System Based on the Escherichia coli rhaBAD Promoter.Biotechnology and Bioengineering, 73: 95-103 (2001)), DeLisa et al.,1999 (DeLisa, M. P., Li, J. C., Rao, G., Weigand, W. A., and Bentley, W.E.: Monitoring GFP-operon fusion protein expression during high celldensity cultivation of Escherichia coli using an on-line optical sensor.Biotechnology and Bioengineering, 65: 54-64. (1999)) or equivalent.However, supplementation of the medium with amino acids like isoleucine,leucine, lysine, methionine, phenylalanine, threonine, tryptophan andvalin or complex media components such as soy peptone or yeast extractmay be beneficial. The process for fermentation is performed in afed-batch mode. Conditions: Temperature 30-40° C., pH 6-7.5, dissolvedoxygen is kept above 20%. After consumption of the initial C-source theculture is fed with the feed media stated above (or equivalent). When adry cell weight in the fermenter of 40 to 90 g/L is reached the cultureis induced with an appropriate inducer corresponding to the usedpromoter system (e.g. IPTG, lactose, arabinose). The induction caneither be performed as a pulsed full induction or as a partial inductionby feeding the respective inducer into the fermenter over a prolongedtime. The production phase should last 4 hours at least. The cells arerecovered by centrifugation in bowl centrifuges, tubular bowlcentrifuges or disc stack centrifuges, the culture supernatant isdiscarded.

13.2 Purification:

The E. coli cell mass is resuspended in 6- to 8-fold amount of lysisbuffer (phosphate or Tris buffer, pH 7-8.5). Cell lysis is preferablyperformed by high pressure homogenization followed by removing of thecell debris by centrifugation in bowl, tubular bowl or disc stackcentrifuges. Supernatant containing the target protein is optionallyfiltrated using a 0.22-10 μm filter and separated via cation exchangechromatography (e.g. Toyopearl MegaCap® II SP-550EC, Toyopearl GigaCapS-650M, SP Sepharose BB, SP Sepharose FF or S HyperCel™) at pH 7-8.5.Elution is performed by a linear increasing NaCl gradient at pH 7-8.5.Fractions containing the target protein are pooled and subsequentlyincubated with 5-10 mM DTT in order to prevent dimerization oraggregation mediated by free cysteine residues. After further additionof 0.8-1 M ammonium sulfate or 2-3 M NaCl, solution is separated viahydrophilic interaction chromatography (e.g. Phenyl Sepharose HP, PhenylSepharose FF, Butyl Sepharose HP, Butyl Sephrose FF, Butyl Toyopearl 650(S,M,C), Phenyl Toyopearl 650 (S,M,C)) at pH 7-8.5. Elution is carriedout at pH 7-8.5 by a linear decreasing ammonium sulfate or NaCl gradientin presence of 5 mM DTT. Fractions containing the target protein with apurity level of minimally 90% are pooled and desalted by diafiltrationin presence of 5 mM DTT followed by concentration to approximately 5mg/ml. Subsequent refolding is performed by diluting the proteinsolution 1:5-1:20 with 50 mM Tris, 150 mM NaCl, 4 mM Cystamin, 10 mMCHAPS at pH 8.5 to a final protein concentration of 0.25-1 mg/ml.Refolding solution is incubated under stirring for 12-36 h at roomtemperature and then separated by cation exchange chromatography (e.g.SP Sepharose FF, SP Sepharose HP, Toyopearl SP-650 (S, M, C)) at pH7-8.5. Elution is performed by a linear increasing NaCl gradient at pH7-8.5. Fractions containing monomeric target protein are pooled andactivated for PEGylation by addition of reducing agents such as DTT, DTEor TCEP. The solution is subsequently incubated at room temperature for2 hours. After diafiltration against Na-phosphate buffer pH 6.5-7.5 or20 mM HEPES buffer pH 6.5-7.5 or Tris buffer pH 8.0 and concentration to5-10 mg/ml, 40-kDa maleimide-polyethyleneglycole (PEG) is added (proteinto PEG ratio of 1:2-1:10). Solution is incubated under stirring for 3-18h at room temperature and subsequently filtrated using a 0.22 μm filter.PEGylated target protein is separated from free PEG and non-PEGylatedtarget protein via cation exchange chromatography (SP Sepharose HP,Toyopearl SP 650M, MacroCap™SP, Source™30S or Fractogel®EMD (M)) at pH5-7. Elution is performed by a linear increasing NaCl gradient.Fractions containing mono-PEGylated target are pooled and formulated in25 mM Na-phosphate, 220 mM endotoxin free trehalose, pH 7.5 viadiafiltration.

Example 14 Pharmaceutical Formulation and Use

14.1 Pharmaceutical Formulation:

Any of the humanized biparatopic polypeptide constructs of theinvention, such as ABII314 to Ab11323, can be selected for themanufacture of a pharmaceutical formulation for subcutaneous applicationhaving a composition as follows:

Drug substance: 100 mg/ml (1 to 3 nmol/ml)

Phosphate buffer: 25 mM

Trehalose: 220 mM

Tween-20: 0.02

Drug substance is formulated in a solution having the above composition,sterilized and stored at 2 to 8° C.

14.2 Pharmaceutical Use:

The solution as prepared under 14.1 above is applied to a patient inneed thereof, such as a human being suffering from AD, by subcutaneousinjection into the belly in a volume of 1 to 2 ml (dosage of 100 to 200mg) every two to four weeks.

The invention claimed is:
 1. A method for treating a disease or disorderthat is associated with A-beta deposition, wherein said method comprisesadministering to a subject in need thereof a pharmaceutically activeamount of at least one polypeptide, wherein said polypeptide comprises afirst immunoglobulin single variable domain and a second immunoglobulinsingle variable domain, wherein the CDR sequences of said firstimmunoglobulin variable domain (CDR(1) sequences) and the CDR sequencesof said second immunoglobulin variable domain (CDR(2) sequences) aredefined as follows: CDR(1)1: SEQ ID NO:17 CDR(1)2: SEQ ID NO:18 CDR(1)3:SEQ ID NO:19 CDR(2)1: SEQ ID NO:14 CDR(2)2: SEQ ID NO:15 CDR(2)3: SEQ IDNO:16, or CDR(1)1: SEQ ID NO:14 CDR(1)2: SEQ ID NO:15 CDR(1)3: SEQ IDNO:16 CDR(2)1: SEQ ID NO:17 CDR(2)2: SEQ ID NO:18 CDR(2)3: SEQ ID NO:19.2. A method of reducing A-beta levels in a body fluid comprisingadministering to a patient in need thereof a polypeptide, wherein saidpolypeptide comprises a first immunoglobulin single variable domain anda second immunoglobulin single variable domain, wherein the CDRsequences of said first immunoglobulin variable domain (CDR(1)sequences) and the CDR sequences of said second immunoglobulin variabledomain (CDR(2) sequences) are defined as follows: CDR(1)1: SEQ ID NO:17CDR(1)2: SEQ ID NO:18 CDR(1)3: SEQ ID NO:19 CDR(2)1: SEQ ID NO:14CDR(2)2: SEQ ID NO:15 CDR(2)3: SEQ ID NO:16, or CDR(1)1: SEQ ID NO:14CDR(1)2: SEQ ID NO:15 CDR(1)3: SEQ ID NO:16 CDR(2)1: SEQ ID NO:17CDR(2)2: SEQ ID NO:18 CDR(2)3: SEQ ID NO:19.
 3. A method for thetreatment or alleviation of a disease, disorder or condition associatedwith A-beta deposition in a human being, comprising the administrationof a polypeptide to the human being in need of such therapy, whereinsaid polypeptide comprises a first immunoglobulin single variable domainand a second immunoglobulin single variable domain, wherein the CDRsequences of said first immunoglobulin variable domain (CDR(1)sequences) and the CDR sequences of said second immunoglobulin variabledomain (CDR(2) sequences) are defined as follows: CDR(1)1: SEQ ID NO:17CDR(1)2: SEQ ID NO:18 CDR(1)3: SEQ ID NO:19 CDR(2)1: SEQ ID NO:14CDR(2)2: SEQ ID NO:15 CDR(2)3: SEQ ID NO:16, or CDR(1)1: SEQ ID NO:14CDR(1)2: SEQ ID NO:15 CDR(1)3: SEQ ID NO:16 CDR(2)1: SEQ ID NO:17CDR(2)2: SEQ ID NO:18 CDR(2)3: SEQ ID NO:19.
 4. The method of claim 3,wherein said disease, disorder or condition is selected from the groupconsisting of Alzheimer's disease, dementia of the Alzheimer type, dryage-related macular degeneration (AMD), glaucoma, cerebral amyloidangiopathy, trisomy 21 (Down's Syndrome), hereditary cerebral hemorrhagewith amyloidosis of the Dutch-type (HCHWA-D), dementia with Lewy Bodies,and sporadic inclusion body myositis.
 5. The method of claim 3, furthercomprising administering an additional therapeutic agent selected fromthe group consisting of ELND-005, Caprospinol, NRM-8499, PBT-2,Posiphen, EHT-0202, CTS-21166, Semagacestat, BMS-708163, BMS-299897,BMS-433796, ELND-006, ELN-475516, ELN-318463, ELN-475513, Begacestat,E-2012, CHF-5074, Dimebolin, and PF-4494700.